tag:blogger.com,1999:blog-23257117574384597482024-03-05T23:21:31.821-08:00Stringent ResponseHere I muse about stuff directly or not-so-directly related to what we do in my lab.
There are two types of posts: streamlined ones for researchblogging.org and not-so-streamlined ones for brain dump. I use labels - do take advantage of that!Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.comBlogger77125tag:blogger.com,1999:blog-2325711757438459748.post-36554312687693699002015-02-09T02:17:00.001-08:002015-02-09T02:23:55.116-08:00Postdoctoral position, 24 months with a possibility of extension <div dir="ltr" style="text-align: left;" trbidi="on">
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<span style="font-family: Calibri; font-size: 14.0pt; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-bidi-font-weight: bold;">The
Laboratory for Molecular Infection Medicine Sweden (MIMS) within the Nordic
EMBL Partnership for Molecular Medicine</span><span style="font-family: Calibri; font-size: 14.0pt; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: Arial; mso-text-raise: -.5pt; position: relative; top: .5pt;">,<span style="letter-spacing: -.4pt;"> </span>Umeå University, Sweden<span style="letter-spacing: 1.05pt;"> </span></span><span style="font-size: 14.0pt; mso-ansi-language: EN-US;"><o:p></o:p></span></h1>
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<b><span style="font-family: Calibri; font-size: 12.0pt; mso-ansi-language: EN-US; mso-bidi-font-family: Arial;">Project description:<o:p></o:p></span></b></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">The focus of the project is
biochemical analysis of </span><i style="mso-bidi-font-style: normal;"><span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial;">Bacillus
subtilis</span></i><span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial;"> ribosome-associated stress factors. The project
addresses questions generated through bioinformatic analysis of ribosomal
factors, and will comprise of a biochemical research programme backed up by a
complementary set of microbiological and structural investigations. The
position is co-financed by funds from Ragnar Söderberg and Carl Tryggers
foundations.<o:p></o:p></span></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial;">Our
laboratory uses a combination of experimental (in vitro biochemistry in a
reconstituted translational system, NGS-based techniques for in vivo
biochemistry, as well as microbiological approaches) and in silico (molecular
evolution) approaches. We are currently supported by the Swedish Research
Council, Ragnar Söderberg foundation, Kempe foundation, Carl Tryggers
foundation, as well as Umeå University funds.<o:p></o:p></span></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">For
further information please contact Dr. Vasili Hauryliuk, </span><span style="mso-ansi-language: EN-US;"><a href="mailto:vasili.hauryliuk@umu.se"><span style="font-family: Calibri; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">vasili.hauryliuk@umu.se</span></a></span><span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";"> or Dr. Gemma C. Atkinson, </span><span style="mso-ansi-language: EN-US;"><a href="mailto:gemma.atkinson@umu.se"><span style="font-family: Calibri; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">gemma.atkinson@umu.se</span></a></span><span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">. See also our <a href="http://www.mims.umu.se/groups/vasili-hauryliuk.html" target="_blank">web page</a>.<o:p></o:p></span></div>
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<b style="mso-bidi-font-weight: normal;"><span style="font-family: Calibri; font-size: 12.0pt; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">A successful candidate
should meet the following criteria:<o:p></o:p></span></b></div>
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<b style="mso-bidi-font-weight: normal;"><span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">Essential:<o:p></o:p></span></b></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">-
Documented proficiency in biochemical assays<o:p></o:p></span></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">- Strong
skills in experimental design and interpretation<o:p></o:p></span></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">- A high
level of proficiency in written and spoken English, as well as scientific
writing<o:p></o:p></span></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">- A
strong publication record in international, peer reviewed journals<o:p></o:p></span></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">- An
ability to work effectively independently and as a group<o:p></o:p></span></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">- A willingness
to learn new techniques<o:p></o:p></span></div>
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<b style="mso-bidi-font-weight: normal;"><span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">Highly desired:<o:p></o:p></span></b></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">-
Experience in ribosomal biochemistry / RNA biochemistry<o:p></o:p></span></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">-
Experience in bacterial genetics, particularly <i style="mso-bidi-font-style: normal;">Bacillus subtilis</i><o:p></o:p></span></div>
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<span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">Applications
should be written in English, comprise of a cover letter, CV with a publication
list and contact information of at least two referees. Documents should be in
MS Word or PDF format and submitted electronically to Dr. Vasili Hauryliuk, </span><span style="mso-ansi-language: EN-US;"><a href="mailto:vasili.hauryliuk@umu.se"><span style="font-family: Calibri; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">vasili.hauryliuk@umu.se</span></a></span><span class="MsoHyperlink"><span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">,</span></span><span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";"> or Dr. Gemma Atkinson, </span><span style="mso-ansi-language: EN-US;"><a href="mailto:gemma.atkinson@umu.se"><span style="font-family: Calibri; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">gemma.atkinson@umu.se</span></a></span><span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial; mso-fareast-font-family: "Times New Roman";">. </span><span style="font-family: Calibri; mso-ansi-language: EN-US; mso-bidi-font-family: Arial;">The
position is available starting spring 2015 (negotiable). <span style="mso-bidi-font-weight: bold;"><o:p></o:p></span></span></div>
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Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com5tag:blogger.com,1999:blog-2325711757438459748.post-60623224866580004342013-03-30T03:30:00.001-07:002013-03-30T03:30:48.518-07:00Two PhD and one postdoctoral position at MIMS<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Arial, Helvetica, sans-serif;">Our lab is branching out. This fall we cross the Baltic sea and set up a new lab in <a href="http://en.wikipedia.org/wiki/Ume%C3%A5">Umeå</a>, Sweden - at the Laboratory for Molecular Infection Medicine Sweden, <a href="http://www.mims.umu.se/">MIMS</a>. Do you like ribosomes? Do you also think that is ppGpp the main G nucleotide in bacteria? Are you stringent enough? Do you know somebody who fits the bill?</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br />
If yes, then good news are that we have two PhD and one postdoctoral positions up for grabs. Drop me a line at vasili.hauryliuk@molbiol.umu.se!</span></div>
Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com2tag:blogger.com,1999:blog-2325711757438459748.post-26816887993274551082013-02-14T06:29:00.002-08:002013-02-14T06:29:35.765-08:00Tet(O) cryoEM structure is out!<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Arial, Helvetica, sans-serif;">So it is finally out: the cryoEM structure of <a href="http://www.nature.com/ncomms/journal/v4/n2/full/ncomms2470.html">Tet(O) on the ribosome</a> we have collaborated on with <a href="http://franklab.cpmc.columbia.edu/franklab/">Joachim Frank's lab</a> is finally published on in Nature Communications. Tet(O) is a bacterial translational GTPase that clears the ribosome from tetracycline antibiotic, and structural data provided in the paper shed light on the mechanism of Tet(O)-mediated resistance. </span><span style="font-family: Arial, Helvetica, sans-serif;">Recently cryoEM of Tet(O)'s close relative, </span><a href="http://www.pnas.org/content/109/42/16900.long" style="font-family: Arial, Helvetica, sans-serif;">Tet(M)</a><span style="font-family: Arial, Helvetica, sans-serif;"> was </span><span style="font-family: Arial, Helvetica, sans-serif;">published</span><span style="font-family: Arial, Helvetica, sans-serif;"> by <a href="http://www.beckmann.genzentrum.lmu.de/">Beckmann</a> and <a href="http://www.wilson.genzentrum.lmu.de/?style=0">Wilson</a> labs, so now one can compare the two. And yes, they look very similar. No surprise there.</span><div>
<div>
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
<div>
<span style="font-family: Arial, Helvetica, sans-serif;"><b>References:</b></span></div>
<div>
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
<div>
<span style="font-family: Arial, Helvetica, sans-serif;">Li et al. Nature Communications (2013) PIMD: <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=23403578">23403578</a></span></div>
<div>
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
<div>
<span style="font-family: Arial, Helvetica, sans-serif;">Dönhöfer at al. PNAS (2012) PIMD: </span><span style="font-family: Arial, Helvetica, sans-serif;"><a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=23027944">23027944</a></span></div>
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<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
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Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com1tag:blogger.com,1999:blog-2325711757438459748.post-65661466641424688252013-01-29T06:12:00.000-08:002013-01-29T06:16:04.982-08:00ppGpp biosyntehsis pathway summed up nicely<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Helvetica Neue, Arial, Helvetica, sans-serif;">A very nice scheme of the ppGpp biosynthesis pathways can be found on the <a href="http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&object=PPGPPMET-PWY&detail-level=2">MetaCyc</a> (<a href="http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=22102576">1</a>). Seems to be a very useful database in general.</span><br />
<span style="font-family: Helvetica Neue, Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Helvetica Neue, Arial, Helvetica, sans-serif;"><b>References:</b></span><br />
<span style="font-family: Helvetica Neue, Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Helvetica Neue, Arial, Helvetica, sans-serif;">Caspi et al. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. NAR 2012 Jan;40(Database issue):D742-53 PIMD: <a href="http://www.ncbi.nlm.nih.gov/pubmed/22102576">22102576</a></span><br />
<span style="font-family: Helvetica Neue, Arial, Helvetica, sans-serif;"><br /></span></div>
Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com1tag:blogger.com,1999:blog-2325711757438459748.post-91897165140354403522012-12-19T02:18:00.003-08:002012-12-19T02:46:37.860-08:00RiboCOURSE Spring 2013: From Ribosome structure to bacterial physiology<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Arial, Helvetica, sans-serif;">An advanced course on bacterial translation and physiology is to be taught at the <a href="http://www.ribocore.uu.se/">RiboCORE</a>, Uppsala University in spring 2013. I will be giving a lecture on the stringent response. You can find the course programme <a href="http://www.ribocore.uu.se/digitalAssets/142/142249_ribocourseplan.pdf">here</a>. </span><span style="font-family: Arial, Helvetica, sans-serif;"><a href="http://onlinelibrary.wiley.com/doi/10.1002/9780470015902.a0023959/abstract">Review on the stringent response</a> written by Gem Atkinson and myself is a nice introduction to the subject.</span></div>
Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com2tag:blogger.com,1999:blog-2325711757438459748.post-5178120700097791552012-11-15T06:58:00.000-08:002012-11-15T07:08:05.479-08:00GDP and SRL don't mix<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Arial, Helvetica, sans-serif;">Translational GTPases run the ribosomal cycle, and the ribosome talks back - it recruits the GTPases when it is a certain state, affects trGTPase's affinity to G nucleotides and activates the GTP hydrolysis when needed. Using Isothermal Titration Calorimetry we showed that <a href="http://www.nature.com/srep/2012/121113/srep00843/full/srep00843.html">binding of GDP nucleotide and of SRL rRNA element to translational GTPases IF2 and EF-G are mutually exclusive</a>. This suggests a neat mechanism for the destabilisation of the ribosome-bound GDP form of the GTPase: the moor has done his duty, the moor can go.</span><br />
<br />
<span style="font-family: Arial, Helvetica, sans-serif;">Due to the technical limitations, the ITC experiments were performed with a 27-nucleotide long RNA piece mimicking the rRNA element as a model. In order to place our results in the framework of the ribosomal cycle we need experiments with the whole ribosome. </span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;"><b>References:</b></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">Mitkevich et al., Scientific Reports 2012 2:843, PIMD: <a href="http://www.ncbi.nlm.nih.gov/pubmed?term=23150791">32150791</a></span></div>
Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com2tag:blogger.com,1999:blog-2325711757438459748.post-22220233552843594152012-11-07T04:47:00.000-08:002012-11-08T05:48:05.414-08:00First PhD defence in the lab<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Arial, Helvetica, sans-serif;">Our first PhD defence took place on November 2d, 2012. Viktoriya Shyp has defended her work "<a href="http://www.ut.ee/en/events/viktoriya-shyp-g-nucleotide-regulation-translational-gtpases-and-stringent-response-factor">G nucleotide regulation of translational GTPases and the stringent response factor RelA</a>". Mike Cashel, the <a href="http://www.nature.com/nature/journal/v221/n5183/abs/221838a0.html">discoverer of ppGpp</a>, served as opponent.</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">Hurray to Vika!</span></div>
Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com4tag:blogger.com,1999:blog-2325711757438459748.post-31124107012259062492012-10-03T01:40:00.001-07:002012-10-03T01:48:58.337-07:00Relacin - a novel antibacterial targeting the stringent response, maybe<div dir="ltr" style="text-align: left;" trbidi="on">
<div style="text-align: justify;">
<span style="font-family: Arial, Helvetica, sans-serif;">The stringent response is a promising target for novel antibacterials: it is involved in virulence and antibiotic insensitivity, and inhibiting the stringent response would disarm the bug, making is both less evil and easier to kill.</span></div>
<br />
<div style="text-align: justify;">
<span style="font-family: Arial, Helvetica, sans-serif;">A new study is came out in PLoS Pathogens describing a novel Rel inhibitor, </span><a href="http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002925" style="font-family: Arial, Helvetica, sans-serif;">relacin</a><span style="font-family: Arial, Helvetica, sans-serif;"> (</span><b style="font-family: Arial, Helvetica, sans-serif;">Fig. 1</b><span style="font-family: Arial, Helvetica, sans-serif;">)</span><span style="font-family: Arial, Helvetica, sans-serif;">. Wexselblatt and colleagues are following up their earlier work on derivatizing ppGpp into a </span><a href="http://www.sciencedirect.com/science/article/pii/S0968089610003767" style="font-family: Arial, Helvetica, sans-serif;">Rel inhibitor</a><span style="font-family: Arial, Helvetica, sans-serif;"> and are now testing the compound not only in vitro, but also in vivo.</span></div>
<div style="text-align: justify;">
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
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<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgJ3DCYc3WnraczUgDeTi4MLW5Kfm-0pekLq-SSGzH03VF9OkxqUwTQlpEjLwB1nDEPznppw2s9ZeCpmmlUGJSCYTLdFirFNKJU3Eb17wgVVqbG_LGhp5gL9vrlCaBV6nj1gKfa-6k1GE4/s1600/Screen+Shot+2012-10-03+at+11.46.19+AM.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="160" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgJ3DCYc3WnraczUgDeTi4MLW5Kfm-0pekLq-SSGzH03VF9OkxqUwTQlpEjLwB1nDEPznppw2s9ZeCpmmlUGJSCYTLdFirFNKJU3Eb17wgVVqbG_LGhp5gL9vrlCaBV6nj1gKfa-6k1GE4/s320/Screen+Shot+2012-10-03+at+11.46.19+AM.png" width="320" /></a></div>
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<div class="separator" style="clear: both; text-align: justify;">
<span style="font-family: Arial, Helvetica, sans-serif; font-size: x-small;"><b>Fig 1: </b>the chemical structure of relacin.</span></div>
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
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<div style="text-align: justify;">
<span style="font-family: Arial, Helvetica, sans-serif;"><span style="font-family: Arial, Helvetica, sans-serif;">They show that relacin efficiently inhibits sporulation of <i>Bacillus subtilis</i>. Sporulation in this organism is driven by <a href="http://jb.asm.org/content/146/2/605.short">ppGpp</a>, and inhibitory effect of relacin is a strong indication that it actually works. </span><span style="font-family: Arial, Helvetica, sans-serif;">However, really high concentrations are needed to achieve significant effects: 0.5 - 2 mM. At these concentrations one would expect that in addition to hitting RelA, relacin will affect all the other ppGpp targets, i.e. </span><a href="http://stringentresponse.blogspot.com/2012/09/one-more-gtpase-that-binds-ppgpp-rf3.html">translational GTPases</a><span style="font-family: Arial, Helvetica, sans-serif;">, </span><a href="http://stringentresponse.blogspot.com/2012/09/ppgpp-regulates-gtp-synthesis-by.html">GTP biosynthesis enzymes </a><span style="font-family: Arial, Helvetica, sans-serif;">etc. The authors do not test these effects. It would be easy to do it in an in vitro translational lysate... but, </span><span style="font-family: Arial, Helvetica, sans-serif;">unfortunately</span><span style="font-family: Arial, Helvetica, sans-serif;">, this is not done. </span><span style="font-family: Arial, Helvetica, sans-serif;">By using a GFP-fusion reporter, they do show that relacin inhibits translation of mid-sporulation protein SpoIIQ, but they do not check that it does not inhibit translation in general. A simple test of GFP expression would do.</span></span></div>
<div style="text-align: justify;">
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
<div style="text-align: justify;">
<span style="font-family: Arial, Helvetica, sans-serif;">With this (potential) absence of specificity relacin is unlikely to be the 'magic bullet' inhibiting just the stringent response and making bacterial less pathogenic, but still viable. However, relacin is just the first step. There is a hope that the derivatives to come will work at more in vivo-relevant concentrations and will be highly Rel-specific.</span></div>
<div style="text-align: justify;">
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
<div style="text-align: justify;">
<b style="font-family: Arial, Helvetica, sans-serif;">References:</b></div>
<br />
<div style="text-align: justify;">
<span style="font-family: Arial, Helvetica, sans-serif;">Wexselblatt et al. Biomed Org Chem (2010) PIMD: </span><a href="http://www.ncbi.nlm.nih.gov/pubmed/20483622" style="font-family: Arial, Helvetica, sans-serif;">20483622</a></div>
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<div style="text-align: justify;">
<a href="http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002925"><span style="font-family: Arial, Helvetica, sans-serif;">Wexselblatt</span><span style="font-family: Arial, Helvetica, sans-serif;"> et al. PLoS Pathogens (2012) </span></a></div>
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Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-38797851404994153352012-09-14T01:01:00.000-07:002012-09-14T02:00:47.067-07:00ppGpp regulates GTP synthesis by inhibiting Gmk and HprT<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Arial, Helvetica, sans-serif;">Another role for ppGpp was discovered by Allison Kriel and colleagues: it <a href="http://www.cell.com/molecular-cell/abstract/S1097-2765%2812%2900696-X?utm_source=ECE001&utm_campaign=&utm_content=&utm_medium=email&bid=3DY2V3F:5MM371F">directly regulates GTP levels by interfering with GTP biosynthetic pathway</a>. It is worth saying that inhibitory effects of </span><span style="font-family: Arial, Helvetica, sans-serif;">(p)</span><span style="font-family: Arial, Helvetica, sans-serif;">ppGpp on IMP dehydrogenase and anenylosuccinate synthetase, first enzymes of the guanylate and adenylate pathways, respectively, were discovered by Gallant and colleagues as early as in </span><a href="http://www.jbc.org/content/246/18/5812.full.pdf" style="font-family: Arial, Helvetica, sans-serif;">1971</a><span style="font-family: Arial, Helvetica, sans-serif;">, so the connection between </span><span style="font-family: Arial, Helvetica, sans-serif;">(p)</span><span style="font-family: Arial, Helvetica, sans-serif;">ppGpp and metabolism of G nucleotides was known long ago.</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">The cool thing about the paper is the approach they use. There can be loads of potential targets of ppGpp, and loads of proteins will be inhibited by it in vitro. Which ones are the relevant ones? Kriel and colleagues usa a top-down approach: first they get a birds-eye view of starvation by running metabolic and transcriptomic analysis of starved wt and ppGpp<sup>0</sup> (i.e. devoid of ppGpp) strains, then identify the ppGpp targets by clustering and pathway analyses, and then follow their predictions up in vitro and an vivo. This is a really powerful approach.</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">The net result is that (p)ppGpp inhibits several enzymes in the GTP biosynthesis pathway, and by doing quantitative experiments, Kriel et al. identify the primary targets, Gmk and HprT </span><span style="font-family: Arial, Helvetica, sans-serif;">(</span><b style="font-family: Arial, Helvetica, sans-serif;">Fig. 1</b><span style="font-family: Arial, Helvetica, sans-serif;">)</span><span style="font-family: Arial, Helvetica, sans-serif;">. All the experiments are done in </span><i style="font-family: Arial, Helvetica, sans-serif;">B. subtilis</i><span style="font-family: Arial, Helvetica, sans-serif;">, and in this bug pppGpp is the major magic spot nucleotide. It is made of GTP, so inhibition of GTP synthesis results in a negative feedback control loop. This loop turns out to be a key component for the control of the GTP levels in the cell.</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">In </span><span style="font-family: Arial, Helvetica, sans-serif;"> </span><i style="font-family: Arial, Helvetica, sans-serif;">B. subtilis</i><span style="font-family: Arial, Helvetica, sans-serif;">, unlike <i>E. coli</i>, ppGpp does not regulate RNA polymerase directly: the regulation goes via <a href="http://www.ncbi.nlm.nih.gov/pubmed?term=15496987">effects on the GTP level</a>. And indeed, a genetic screen performed by </span><span style="font-family: Arial, Helvetica, sans-serif;">Kriel et al.</span><span style="font-family: Arial, Helvetica, sans-serif;"> showed that regulation of GTP metabolises by ppGpp, not of polymerase is crucial for the bacterial survival under stress. The have found 37 suppressor mutations leading to survival of the </span><span style="font-family: Arial, Helvetica, sans-serif;">ppGpp</span><sup style="font-family: Arial, Helvetica, sans-serif;">0</sup><span style="font-family: Arial, Helvetica, sans-serif;"> strain - and most of these turned out to be in the GTP biosynthesis pathway. In the <i>E. coli</i> case,<a href="http://www.ncbi.nlm.nih.gov/pubmed/20946586"> the supressor mutations are usually in the RNA polymerase</a>.</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;"> The negative control of GTP synthesis by ppGpp turned out to be crucial for bacterial well-being - in the </span><span style="font-family: Arial, Helvetica, sans-serif;">ppGpp</span><sup style="font-family: Arial, Helvetica, sans-serif;">0</sup><span style="font-family: Arial, Helvetica, sans-serif;"> cells high levels of GTP caused cell death, though the mechanism is still unclear (<b>Fig. 1</b>). </span><span style="font-family: Arial, Helvetica, sans-serif;">Kriel et al.</span><span style="font-family: Arial, Helvetica, sans-serif;"> proposed several possible explanations: inhibition with ATP-consuming enzymes, excessive up-regulation of the rRNA transcription, effects on dGTP synthesis etc.</span><br />
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<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgCbnvZzHnIxVu2I3Jz7Q406snO05ATL5Lg54FviAa9VrG2P4qgBCzMV7IhPBX_aQpYjdnnQoc-BcOKXvyfiWRI3PzvPophoR_geSbWcTzWDJETHIGLmv72xFxF9hgN_gJzUwN3A2WYehw/s1600/Screen+Shot+2012-09-14+at+11.57.19+AM.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgCbnvZzHnIxVu2I3Jz7Q406snO05ATL5Lg54FviAa9VrG2P4qgBCzMV7IhPBX_aQpYjdnnQoc-BcOKXvyfiWRI3PzvPophoR_geSbWcTzWDJETHIGLmv72xFxF9hgN_gJzUwN3A2WYehw/s320/Screen+Shot+2012-09-14+at+11.57.19+AM.png" width="296" /></a></div>
<span style="font-size: x-small;"><span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;"><b>Fig. 1:</b> ppGpp's role in <i>B. subtilis</i> survival via regulation of GTP biosynthesis. Figure from </span><span style="font-family: Arial, Helvetica, sans-serif;"><a href="http://www.cell.com/molecular-cell/abstract/S1097-2765%2812%2900696-X?utm_source=ECE001&utm_campaign=&utm_content=&utm_medium=email&bid=3DY2V3F:5MM371F">Kriel et al</a>.</span></span><br />
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<b><span style="font-family: Arial, Helvetica, sans-serif;">References:</span></b><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<a href="http://www.cell.com/molecular-cell/abstract/S1097-2765%2812%2900696-X?utm_source=ECE001&utm_campaign=&utm_content=&utm_medium=email&bid=3DY2V3F:5MM371F"><span style="font-family: Arial, Helvetica, sans-serif;">Kriel et al, Cell (2012) in press</span></a><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">Gallant et al. JBC (1971), 246 (18): 5812-5816, PIMD: <span style="background-color: white; color: #575757; line-height: 16.79166603088379px; white-space: nowrap;"><a href="http://www.ncbi.nlm.nih.gov/pubmed/4938039">4938039</a></span></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">Krasny and Grouse, EMBO J. (2004), 23(22): </span><span style="background-color: white; font-family: arial, helvetica, clean, sans-serif; line-height: 15.944443702697754px;">4473-83, </span><span style="font-family: Arial, Helvetica, sans-serif;">PIMD: </span><span style="background-color: white; color: #575757; font-family: arial, helvetica, clean, sans-serif; line-height: 15.402777671813965px; white-space: nowrap;"><a href="http://www.ncbi.nlm.nih.gov/pubmed?term=15496987">15496987</a></span><br />
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<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-59390457106624510232012-09-13T05:52:00.004-07:002012-09-13T13:30:02.914-07:00One more GTPase that binds ppGpp: RF3<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Arial, Helvetica, sans-serif;">The stringent response alarmone ppGpp is very, very similar to GDP: add two more phosphates and you have it... So it is only natural that GTPases mistake the two.</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">Now Kihira and collegues has added <a href="http://www.sciencedirect.com/science/article/pii/S0014579312006862#">one more</a> to the list - bacterial termination factor 3, RF3. This factor is involved in release of the class one release factors, RF1 and RF2. It binds to the ribosome in the GDP-bound state, <a href="http://www.ncbi.nlm.nih.gov/pubmed/11595190">exchanges GDP to GTP</a> and kicks<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1170035/"> the class 1 factors off</a>. </span><span style="font-family: Arial, Helvetica, sans-serif;">ppGpp:RF3 complex is not as active as GDP:RF3 one, a situation similar to that with <a href="http://www.ncbi.nlm.nih.gov/pubmed/20713063">initiation factor 2, IF2</a>. </span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">The question is is it a specific regulatory mechanism or are GTPases just promiscuous enough? After all, even archael elongation factor 1A, <a href="http://www.springerlink.com/content/j865ur44g13876l1/">aEF1A, is inhibited by ppGpp</a> as well. And these two never meet in nature...</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">Kanjee and collegues have recently compiled a <a href="http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2012.08177.x/full">list of proteins that ppGpp binds to</a>... or is expected to bind to. GTPases are there, as a whole class of enzymes.</span><br />
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<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;"><b>References:</b></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;"><a href="http://www.sciencedirect.com/science/article/pii/S0014579312006862#">Kihira et al. FEBS J. (2012) in press</a></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">Kanjee et al. Mol. Microbiology (2012): 85 (6) 1029-1043 PIMD: <a href="http://www.ncbi.nlm.nih.gov/pubmed?term=Direct%20binding%20targets%20of%20the%20stringent%20response%20alarmone%20(p)ppGpp">22812515</a></span></div>
Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-7553261910816284442012-07-30T02:45:00.001-07:002012-08-21T00:30:22.475-07:00Positive feedback control of E. coli RelA by its product ppGpp<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Arial, Helvetica, sans-serif;">ppGpp regulates numerous targets, and now we added one more: <a href="http://www.ncbi.nlm.nih.gov/pubmed/22814757">the stringent response factor RelA itself</a>. Using an in vitro stringent response system we showed that ppGpp dramatically increases the turnover rate of RelA, both is the system where RelA is activated by the ribosomes (both naked and programmed with tRNA and mRNA) and in the system where RelA is activated by the ribosomal protein L11 alone.</span><br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjdFYC0q0H8893npQUg5fmrDzlk_hmIXp1FIWf3yrsdxzZSuhEHiSnLRaymXz0X_h14LM6wvi0RG6WJ-jfuwYEe4aFb601b9P6J5xUyapX-UKv2GbDE2lSAsIYHQWvCXurAaFj37KYb-PU/s1600/Screen+Shot+2012-07-30+at+12.31.26+PM.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><span style="font-family: Arial, Helvetica, sans-serif;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjdFYC0q0H8893npQUg5fmrDzlk_hmIXp1FIWf3yrsdxzZSuhEHiSnLRaymXz0X_h14LM6wvi0RG6WJ-jfuwYEe4aFb601b9P6J5xUyapX-UKv2GbDE2lSAsIYHQWvCXurAaFj37KYb-PU/s320/Screen+Shot+2012-07-30+at+12.31.26+PM.png" width="308" /></span></a></div>
<span style="font-family: Arial, Helvetica, sans-serif;"><b style="font-size: small;">Figure 1:</b><span style="font-size: x-small;"> RelA activation in the 70S-driven in vitro system upon addition of ppGpp</span></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">We did 70S and L11 tittrations and demonstrated that ppGpp increases RelA's <i>k<span style="font-size: x-small;">cat</span></i>, making it a more efficient enzyme:</span><br />
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<div class="separator" style="clear: both; text-align: center;">
<span style="font-family: Arial, Helvetica, sans-serif; margin-left: 1em; margin-right: 1em;"><img border="0" height="290" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiczWFti6m8zLYssxHKmfDd_eFWaYo9e79kDeJODmcnZ-zmE7H0FZNlrnllsu-e71xwZkmRk3xi9wse8TwoDZ8RhAAKhKTmT-qSxVtE0kik6SUlRpT8q0RTF5sBpx-m9Yj_9ik9f09cR9g/s320/Screen+Shot+2012-07-30+at+12.34.58+PM.png" width="320" /></span></div>
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<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif; font-size: x-small;"><b>Figure 2.</b> RelA activity as a function of the 70S concentration in presence and absence of ppGpp</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">What next? First off, we do not know where ppGpp binds and how it regulates RelA on the mechanistic level. Second, since there are at least <a href="http://www.ncbi.nlm.nih.gov/pubmed/21858139">30 groups of the RSH proteins</a>, we will figure out which are activated by this mechanism, and which are not. This will provide us some vital clues for understanding the computational properties of the stringent response system. Third, after this in vitro result it is instrumental to show the ppGpp-mediated activation in vivo. </span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">PS: and now our paper got covered as a <a href="http://www.nature.com/nchembio/journal/v8/n9/full/nchembio.1051.html">Research Highlight in Nature Chemical Biology</a>. Yay!</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;"><b>References:</b></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span>
<span style="font-family: Arial, Helvetica, sans-serif;">Shyp et al., EMBO Reports (2012) doi: 10.1038/embor.2012.106.</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;">PIMD: <a href="http://www.ncbi.nlm.nih.gov/pubmed/22814757">22814757</a></span><br />
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Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com2tag:blogger.com,1999:blog-2325711757438459748.post-35316506360546818292012-07-09T06:35:00.002-07:002012-07-10T08:13:43.462-07:00iGEM2011: GFP-based readout for ppGpp concentration in vivo<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Arial, Helvetica, sans-serif;">Measuring ppGpp concentration in individual living cells with good temporal resolution would be great. I've been musing about a possibility of doing that <a href="http://stringentresponse.blogspot.com/2012/03/measuring-nucleotide-concentrations.html">using RNA aptamers</a>, but that's just musing. It seems like am <a href="http://2011.igem.org/Team:NTNU_Trondheim/Team">iGEM team from the University of Trondheim</a> tried setting up a <a href="http://2011.igem.org/Team:NTNU_Trondheim">GFP-based reported system</a>, and this system is, maybe, possibly, probably, working. <a href="http://2011.igem.org/Team:NTNU_Trondheim/Stress_Sensor">Somewhat</a>. </span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span><br />
<span style="background-color: white;"><span style="font-family: Arial, Helvetica, sans-serif;">Unfortunately</span></span><span style="font-family: Arial, Helvetica, sans-serif;"> there is no publication. However, there is a popular article about the whole affair, <a href="http://www.bion.no/filarkiv/2012/02/Bakteriedesign.pdf">in Norwegian</a> and there is a<a href="http://2011.igem.org/Team:NTNU_Trondheim/Stress_Sensor"> short report on the iGEM webpage</a>. </span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;">Apropos to the technical issues that are listed in the original report, such as dramatic leakage of the GFP reporter in the absence of stress stimuli, there are several conceptual concerns. First, the system is based on translation of the GFP reporter during the stringent response, and during the stringent response <a href="http://www.ncbi.nlm.nih.gov/pubmed?term=11946810">translation is strongly inhibited</a>. Second, GFP (they use red version of it, <a href="http://www.nature.com.sci-hub.org/nbt/journal/v22/n12/full/nbt1037.html">mCherry</a>) has to mature in order to become bringt, and that takes some time - from minutes to hours, depending on the conditions and what sort of GFP variant it is. For <a href="http://www.ncbi.nlm.nih.gov/sites/entrez/16299475?dopt=Abstract&holding=f1000,f1000m,isrctn">mCherry maturation time is 15-40 minutes</a>, and this is comparable with <i>E. coli </i>generation time. Therefore, first, one would expect a very pronounced lag before the SR is engaged and the corresponding readout and, second, all the</span><span style="font-family: Arial, Helvetica, sans-serif;"> </span><span style="font-family: Arial, Helvetica, sans-serif;">fluctuations in the ppGpp concentrations happening on the timescale below 10s of minutes</span><span style="font-family: Arial, Helvetica, sans-serif;"> will be </span><span style="font-family: Arial, Helvetica, sans-serif;">averaged</span><span style="font-family: Arial, Helvetica, sans-serif;"> out</span><span style="background-color: white; font-family: Arial, Helvetica, sans-serif;">. Third, GFP is very stable, so this reporter system will have severe memory effects - once the cell has committed to stringency, it will produce GFP, and even though stringency is reversed, GFP will stay. Maybe it is possible to turn this into a </span><a href="http://www.ncbi.nlm.nih.gov/pubmed/18430255" style="background-color: white; font-family: Arial, Helvetica, sans-serif;">feature</a><span style="background-color: white; font-family: Arial, Helvetica, sans-serif;">, but I am not sure how. And, lastly, brightness of the </span><a href="http://stringentresponse.blogspot.com/2010/12/gfp-based-quantification-of-stuff-in.html" style="background-color: white; font-family: Arial, Helvetica, sans-serif;">GFP depends on the pH and redox potential of the environment</a><span style="background-color: white; font-family: Arial, Helvetica, sans-serif;">, and these things change in the stressed cells.</span></div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-86333136104929123242012-07-05T01:29:00.001-07:002012-07-10T02:10:34.951-07:00More fun with fun12... right, that was a dreadful joke<div dir="ltr" style="text-align: left;" trbidi="on">
<div style="text-align: left;">
<span style="font-family: Arial, Helvetica, sans-serif;">I like translational GTPases. I particularly like bacterial ones. Hell, I am titrating one with GTP as we speak, so I am partial. However, eukaryotic translational GTPases are also OK, particularly the ones that have bacterial homologues.</span></div>
<div style="text-align: left;">
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
<div style="text-align: left;">
<span style="font-family: Arial, Helvetica, sans-serif;">One of these is eukaryotic initiation factor 5B, eIF5B, a homologue of bacterial initiation factor 2, IF2. In <i>S. cerevisiae</i> eIF5B is called <a href="http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=YAL035w">Fun12</a>. eIF5B is a GTPase, and translational GTPases are tightly regulated. During translation initiation Fun12 is involved in the <a href="http://www.nature.com/nature/journal/v403/n6767/full/403332a0.html">subunit joining</a>, and GTP hydrolysis is coupled with factor's <a href="http://www.sciencedirect.com/science/article/pii/S0092867402011716">release from the initiation complex</a>. </span></div>
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<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
<div style="text-align: left;">
<span style="font-family: Arial, Helvetica, sans-serif;">Now it turnes out that Fin12/eIF5B has another function outside of translation initiation: together with the 60S subunit it proofreads the ribosomal assembly (<a href="http://www.ncbi.nlm.nih.gov/pubmed?term=22751017">Lebaron et al. 2012</a> and <a href="http://www.ncbi.nlm.nih.gov/pubmed/22770215">Strunk et al. 2012</a>). Assembly of the ribosomal subunits is a multistep process, with r-proteins binding is certain sequence, assisted by various assembly factors, with rRNA being cut and remodeled. One of the steps is cleavage of the 18S rRNA by the Nob1 RNAse. And this stage, apparently, is stimulated by Fun12. Bacterial Fun12 homologue, IF2, is <a href="http://www.sciencedirect.com/science/article/pii/S1097276509003992">GTP-dependently rearranging the ribosomal structure by inducing intersubunit rotation</a>, so the same process is now suggested to play the role in 40S maturation in yeast via driving the ribosome in Nob1-susceptible state.</span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;">In eucaryotes translation-incompetent ribosomes undergo <a href="http://www.ncbi.nlm.nih.gov/pubmed/17188037">so-called nonfunctional rRNA decay, NRD</a>. By linking functionality on Fun12 binding and activation via consecutive 60S binding, translational functionality is linked to rRNA processing, providing another safety net making sure that only functional ribosomes are involved in translation, and the nonfunctional ones are rapidly cleared out.</span></div>
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<span style="font-family: Arial, Helvetica, sans-serif;"><b>References:</b></span></div>
<div style="text-align: left;">
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span><span style="font-family: Arial, Helvetica, sans-serif;">Lebaron et al. <span style="background-color: white;">Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits. Nature Str. Mol. Biol. 2012 <i>in press</i> PIMD: </span><span style="background-color: white; color: #575757; line-height: 14.999999046325684px; white-space: nowrap;"><a href="http://www.ncbi.nlm.nih.gov/pubmed/22751017">22751017</a></span></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;">Strunk et al. <span style="background-color: white;">A Translation-Like Cycle Is a Quality Control Checkpoint for Maturing 40S Ribosome Subunits. Cell </span><span style="background-color: white;">2012 vol. 150 (1) pp. 111-121 PIMD: </span><span style="background-color: white;"><a href="http://www.ncbi.nlm.nih.gov/pubmed/22770215">22770215</a></span></span></div>
</div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-18785720259289069222012-07-03T01:40:00.001-07:002012-07-03T02:28:52.358-07:00One more '-omics' analysis of the stringent response: BIBLIOMICS<div dir="ltr" style="text-align: left;" trbidi="on">
<div style="text-align: left;">
<span style="font-family: Arial, Helvetica, sans-serif;">The stringent response is confusing, no doubt about that. I personally get exceedingly confused when I read <i>in vivo</i> papers from the 80s. I somehow hope that there should be hidden gems there, and it is just my stupidity that stops me from discovering these.... so I try... and I get confused. <i>In vivo</i> data confuse me, and <i>in vivo</i> data from the 80s.... I am lost.</span></div>
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<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span></div>
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<span style="font-family: Arial, Helvetica, sans-serif;">Importantly, I <b>always</b> try to read one paper at a time, maximum ten, <b>rarely</b> more then twenty. Imagine what happens if one would read them <b>all</b>? And how, I wonder, how would one call this sort of thing? Wonder no more; enter <a href="http://www.microbialinformaticsj.com/content/1/1/14">Carneiro and colleagues</a>. What they did, they collected the whole <b>bibliome</b> about the <i>E. coli</i> stringent response and analyzed it in attempt to gain a birds-eye view, providing '<span style="background-color: white;"><i>a more systematic understanding of this cellular response</i>'. </span></span></div>
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<span style="font-family: Arial, Helvetica, sans-serif;"><span style="background-color: white;">They summarize the nitty-gritty of the stringent response in the magnificent<b> Figure 1</b>.</span></span></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhyyZUTnn9mAQPBTOuJ4sHDWf91LQb6So-sBFkj_o0r0zEpaySTQZfPEqvKKrHiRG59LAC6JLA0LJWN3lb0BCWsMNhA-GQzqril0dfFJV_q_SvUcb8pQCUnl-03wlrtCrR_dXp6mBNlCng/s1600/Screen+Shot+2012-07-03+at+11.10.17+AM.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhyyZUTnn9mAQPBTOuJ4sHDWf91LQb6So-sBFkj_o0r0zEpaySTQZfPEqvKKrHiRG59LAC6JLA0LJWN3lb0BCWsMNhA-GQzqril0dfFJV_q_SvUcb8pQCUnl-03wlrtCrR_dXp6mBNlCng/s320/Screen+Shot+2012-07-03+at+11.10.17+AM.png" width="284" /></a></div>
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<span style="font-family: AdvOT07517017; font-size: 10pt;"> </span><span style="font-family: Arial, Helvetica, sans-serif;">The sheer number of mistakes they make is owe-inspiring and truly shows the power of the high-throughput learning. It is OK that tRNAs have their anticodons on one the 3' (or is it 5'?) end and amino acids are attached where the anticodons should be. Fine, that the amino acid moieties are <b>outside</b> of the ribosome when the tRNAs are attached. Fine, that there is no way in hell one can figure out there are the A- , P- and E-sites of their ribosomes, and, surely, it would be <i>nice</i> to be able to know position of at least the A-site when we are talking about the stringent response. But what is truly fabulous, is that the ribosomal protein L11 is part of the <b>small</b> <b>ribosomal subunit</b> (L... the letter <b>L</b> is giving us a hint... <i>large</i>, maybe?...). Since RelA is interacting with L11, so this causes a bit of a <i>trouble</i>, and RelA ends up on the <b>small subunit</b> as well (ah, never mind <a href="http://www.ncbi.nlm.nih.gov/pubmed?term=4595574">this paper</a>). In this awkward position RelA has no chances of inspecting the CCA of the A-site tRNA, but maybe it is for the best, given how messed up the tRNA already is and that we have no idea where the A-site could be...</span><br />
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<span style="font-family: Arial, Helvetica, sans-serif;">The bird-eye view allows Carneiro and collegues to make some information-packed generalizations. I am not sure what we learn this way, but the figures speak for themselves:</span><br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiWVguhlS7x6LSNl5MfeSe_vw0v9nMNXD2rTZoqjU74hoQR3CUhW8ZTCzpMTIF3TDw-9xlWNneiVG1wPWM-RwrKcikVLZDe9SCg_SqvElkl3xUUFpqbGvb65mP2aMsBZHOZxO0Z4BRwX2A/s1600/Screen+Shot+2012-07-03+at+11.23.04+AM.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="222" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiWVguhlS7x6LSNl5MfeSe_vw0v9nMNXD2rTZoqjU74hoQR3CUhW8ZTCzpMTIF3TDw-9xlWNneiVG1wPWM-RwrKcikVLZDe9SCg_SqvElkl3xUUFpqbGvb65mP2aMsBZHOZxO0Z4BRwX2A/s320/Screen+Shot+2012-07-03+at+11.23.04+AM.png" width="320" /></a></div>
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<span style="font-family: Arial, Helvetica, sans-serif;">Rrrright, the dashed line overtook the solid one and they never crossed, therefore the ratios between the blue and red bars changed with time... I want some error-bars, then it will be really, really nice and scientific.</span><br />
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<span style="font-family: Arial, Helvetica, sans-serif;">But it is not only the figures that are great. The text is awesome as well. '<span style="background-color: white;"><i>Later, in 1980, the ppGpp level was found
to be controlled by the SpoT enzyme via GTP hydrolysis
activity (PMID: 6159345)</i>'. GTP hydrolysis! By SpoT! Yay! '</span><span style="background-color: white;"><i>The 50S ribosomal subunit protein L11 has been indirectly implicated in the
feedback inhibition of (p)ppGpp, because ribosomes
lacking this protein are unable to stimulate the synthesis
of these nucleotides (PMID:11673421; PMID:17095013)
[39,61].</i>' Feedback inhibition! This redefines the meaning of feedback... Wow! No wonder L11 migrated on the large subunit... </span></span><br />
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<span style="font-family: Arial, Helvetica, sans-serif;"><span style="background-color: white;">Being subjected to such a monumental degree of confusion, the authors started expressing themselves in a most peculiar way; for the lack of a better word I would call it 'cautious': '</span><span style="background-color: white;"><i>As a result,
it was possible to perceive the relevance of specific
translation GTPases known to be inhibited by (p)ppGpp
nucleotides</i>'. '</span><span style="background-color: white;"><i>Studies showed that (p)ppGpp inhibits
translation by repressing the expression of ribosomal
proteins and also potentially inhibiting the activity of
the particular proteins</i>'. </span></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><span style="background-color: white;"><br /></span></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><span style="background-color: white;">I certainly hope that soon the authors should take on the mighty ribosome. There are many more papers in the ribosomal bibliome, and the level of confusion might be even higher. I would also recommend some 3D plots!</span></span><br />
<br />
<span style="font-family: Arial, Helvetica, sans-serif;"><b> References:</b></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span><br />
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<span style="font-family: Arial, Helvetica, sans-serif;"><span style="background-color: white;">Carneiro, S., Lourenço, A., Ferreira, E. C., & Rocha, I. (2011). Stringent response of Escherichia coli: revisiting the bibliome using literature mining. </span><i style="background-color: white;">Microbial informatics and experimentation</i><span style="background-color: white;">, </span><i style="background-color: white;">1</i><span style="background-color: white;">(1), 14. doi:10.1186/2042-5783-1-14 PIMD: </span><span style="background-color: white; color: #575757; line-height: 15px; white-space: nowrap;"><a href="http://www.ncbi.nlm.nih.gov/pubmed?term=22587779">22587779</a></span></span></div>
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<br /></div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-7358300939567232052012-07-03T00:55:00.002-07:002012-07-03T01:58:23.826-07:00One more crazy little thing<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: Arial, Helvetica, sans-serif;">Stringent response is run by a family of proteins called <a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0023479">RelA-SpoT Homologues, RSH</a>, and these come in two flavors: the long ones and the short ones. The long ones have both ppGpp synthesizing and ppGpp hydrolyzing domains, and either both are active, with synthesizing being the dominant one (that would be the ancestral Rel) or both are active, with hydrolyzing being the dominant one (SpoT) or only the synthesizing one is active (RelA). The short RSHs are more variable. They have only one of the domains, so they can either synthesize (SAS, small alarmone synthetase) or hydrolyze (SAH, small alarmone synthetase) ppGpp. What is fun, is that in addition they can have other domains, sometimes with very peculiar functions.</span><br />
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<span style="font-family: Arial, Helvetica, sans-serif;">A very peculiar SAS was characterized recently by Maya Murdeshwar and Dipankar Chatterji. They call it <a href="http://jb.asm.org/content/early/2012/05/21/JB.00258-12.full.pdf">MS_RHII-RSD</a>, or, using terminology proposed in <a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0023479">our paper with Gemma Atkinson</a>, actRel<span style="font-size: xx-small;">Msm</span>. This SAS from <i>Mycobacterium smegmatis</i> in addition to the ppGpp synthetic activity has another one, quite unexpected. It has a dedicated domain capable of hydrolyzing DNA:RNA duplexes via its RNAse H domain.</span></div>
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<span style="font-family: Arial, Helvetica, sans-serif;">Quite bizarre, quite. </span><br />
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<b><span style="font-family: Arial, Helvetica, sans-serif;">References:</span></b><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><br /></span><br />
<span style="font-family: Arial, Helvetica, sans-serif;">MS_RHII-RSD: a dual function RNase HII - (p)ppGpp synthetase from <i>Mycobacterium smegmatis</i>. M. Murdeshwar and D. Chatterji. J. Bacteriology, 2012, <i>in press </i>PIMD: </span><a href="http://www.ncbi.nlm.nih.gov/pubmed/22636779" style="background-color: white; font-family: Arial, Helvetica, sans-serif; line-height: 15px; white-space: nowrap;">22636779</a><br />
<span style="font-family: Arial, Helvetica, sans-serif;"><span style="background-color: white; color: #575757; font-size: 11px; line-height: 15px;"> </span></span></div>
</div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com1tag:blogger.com,1999:blog-2325711757438459748.post-16437382263196415782012-03-14T01:59:00.000-07:002012-03-14T02:20:07.270-07:00Proline residue in L11 as a key regulator of translational GTPases?<div dir="ltr" style="text-align: left;" trbidi="on">
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The ribosome is run by translational GTPases. Translational GTPases, in their turn, are regulated by the ribosome. They all bind in the same region (GAC, GTPase associated center) of the ribosome. In bacteria the GTPase binding site consists of a couple of rRNA elements: SRL (sarcin-ricin loop) and thiostrepton loop and several ribosomal proteins:L7/L12 stalk (L10 and L7/L12) and L11.<br />
<br />
The latter is the main hero of <a href="http://www.nature.com/nsmb/journal/vaop/ncurrent/full/nsmb.2254.html">a fresh paper</a> in Nature Structural and Molecular Biology by Wang and coworkers. They show that bacterial translational GTPases (such as <a href="http://en.wikipedia.org/wiki/EF-G">EF-G</a>) when binding to the ribosome act as peptidyl-prolyl cis-trans isomerases (<a href="http://en.wikipedia.org/wiki/Prolyl_isomerase">PPIase</a>s) driving isomerisation in the conserved residue in the ribosomal protein L11. This isomerisation, in turn, transmits signal to the ribosomal protein L7 /L12 - something that is necessary for efficient GTPase cycling on the ribosome.<br />
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I like L11 - it is a key protein for stringent response, and without it stringent factor RelA does not work, as was shown using<i> E. coli </i>mutants lacking L11 (<a href="http://jb.asm.org/content/140/2/734.long">Dabbs J. Bac 1979</a>). These mutants are perfectly viable, but grow ten times slower then the wild type <i>E. coli</i>, most probably due to defects in the ribosome assembly (<a href="http://www.jbc.org/content/256/5/2284.long">Hampl et al. JBC 1981</a>). The very viability of the L11 knock-out strains tells us that L11 is not the key for keeping the ribosome running. In fact, less than a half of ribosomal proteins can be knocked-out in <i>E. coli </i>(22 out of 54, <a href="http://www.sciencedirect.com/science/article/pii/S0022283611009934">Shoji et al. JMB 2011</a>), making L11 one of the less-important ones... and keeping an eye of the translational GTPases is definitely not one of the less-important functions!<br />
<br />
This seems to be bit paradoxical - a ribosomal protein that is dispensable involved in something that is very central for protein biosynthesis. It gets even more fascinating when you look at the evolutionary aspect of the story (Gem Atkinson does that in <a href="http://feedproxy.google.com/~r/ProteinEvolution/~3/WifkX1-IZOQ/switches-and-latches-on-ribosome.html">her blog</a>). Wang and colleagues managed to map the PPIase site of EF-G. As they show PPIase activity is universal for all the bacterial translational GTPases they tested, and the PPIase site is, surprisingly, quite a variable region of the G domain! So, do they all reinvent the weel separately? This is all most peculiar.<br />
<br />
<b>References:</b><br />
<br /></div>
Wang et al. A conserved proline switch on the ribosome facilitates the recruitment and binding of trGTPases. <i>Nat Struct Mol Biol</i> (2012) PIMD: <a href="http://www.ncbi.nlm.nih.gov/pubmed/22407015">22407015</a></div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-19669264634827650102012-03-13T07:20:00.002-07:002012-03-16T12:57:31.518-07:00ppGpp induces production of fruiting bodies in Myxococcus xanthus<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="float: left; padding-bottom: 5px; padding-left: 5px; padding-right: 5px; padding-top: 5px;"><a href="http://researchblogging.org/news/?p=3286"><img alt="This post was chosen as an Editor's Selection for ResearchBlogging.org" src="http://www.researchblogging.org/public/citation_icons/rb_editors-selection.png" style="border: 0;" /></a></span>
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<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-family: inherit;"><i>E. coli</i> is boring, admit it. At least in comparison with <span style="font-style: oblique;">Myxococcus xanthus</span>:<a href="http://en.wikipedia.org/wiki/Myxococcus_xanthus"> a self-organized, predatory saprotrophic single-species biofilm called a swarm</a> according to the Wikipedia. Now that sounds exciting! I wish one day somebody would call me "a self-organized, predatory biofilm called a swarm"! That would make a lovely email signature: "Vasili Hauryliuk, PhD, </span> self-organized, predatory biofilm called a swarm". Hell yes.<br />
<br />
<div>
But I digress. Stringent response (or, to be more specific, <a href="http://onlinelibrary.wiley.com/doi/10.1002/9780470015902.a0023959/abstract">RelA-mediated production of alarmone molecule ppGpp</a>) regulates loads of things in bacterial physiology: it turns on bacterial survival mode and <a href="http://stringentresponse.blogspot.com/2011/11/dksa-and-ppgpp-regulate-transcription.html">shuts down production of ribosomes</a>, it <a href="http://stringentresponse.blogspot.com/2011/01/methicillin-resistant-s-aureus-mrsa.html">induces virulence</a> (cornered bacteria are deadly) and makes bugs <a href="http://stringentresponse.blogspot.com/2011/12/active-role-of-stringent-response-in.html">more resistant to antibiotics</a>. Now let us just imagine for a moment what stringent response can do to a "a self-organized, predatory biofilm called a swarm"! Exactly that was investigated in recent paper by Konovalova and colleagues (<a href="http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2012.08020.x/abstract">Konovalova et al. Mol Microbiology 2012</a>).<br />
<br />
Unlike boring <i>E. coli</i>, <span style="font-style: italic;">Myxococcus xanthus </span>has a life cycle (<b>Fig. 1</b>). It can swarm happily gobbling up other bacteria, or, if food supply is low, it can form a fruiting body (a life-stile similar to that of<a href="http://en.wikipedia.org/wiki/Slime_mold"> slime molds </a>who are not bacteria but eucaryotes).<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjOl8esF3wjODzxYOixDXUURKAF1ZwRAKRXRwx0Jv6v6TWBDh4DjNsJq5PriwiZeMQUBFcIahMbl4h2W0C0_VAM0r5O1SANr-YQouvQWXB_cMcyCJTpW-vPVHOU8g2IaRRrrSwrBrJToDc/s1600/Web.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em; text-align: center;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjOl8esF3wjODzxYOixDXUURKAF1ZwRAKRXRwx0Jv6v6TWBDh4DjNsJq5PriwiZeMQUBFcIahMbl4h2W0C0_VAM0r5O1SANr-YQouvQWXB_cMcyCJTpW-vPVHOU8g2IaRRrrSwrBrJToDc/s320/Web.jpg" width="309" /></a></div>
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<b><br class="Apple-interchange-newline" /><span style="font-size: x-small;">Fig. 1. </span></b><span style="font-size: x-small;">Life cycle of a self-organized, predatory biofilm called a swarm (AKA </span><span style="font-size: x-small; font-style: italic;">Myxococcus xanthus</span><span style="font-size: x-small;">).</span><br />
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Formation of the fruiting bodies depends on the functionality of the stringent response system (<a href="http://genesdev.cshlp.org/content/12/7/1022.long">Harris et al. Gens Dev. 1998</a>). How Konovalova and colleagues fill in the molecular details presenting an example of post-translational activation of secretion by regulated proteolysis. Here is how it works.<br />
<br />
Formation of the fruiting bodies depends on the cell-to-cell signaling, and this process, obviously, happens outside of the cell. It involves proteolysis of several extracellular target proteins by a <a href="http://en.wikipedia.org/wiki/Subtilisin">subtilisin</a>-like protease PopC, which needs to be exported outside of the cell in order to do its job. So now it turns out that RelA, working together with PopD protein, regulates <a href="http://www.ncbi.nlm.nih.gov/pubmed/18854146">PopC export</a>, which is activated during starvation (and, therefore, production of ppGpp). The PopD:PolC complex formation is not affected by <a href="http://en.wikipedia.org/wiki/Guanosine_pentaphosphate">ppGpp</a>, suggesting that regulation of export by RelA is using some indirect mechanism. And indeed, PopD turned out to be degraded during starvation in a <a href="http://www.ncbi.nlm.nih.gov/pubmed/19744556">FtsH</a>-dependent manner, releasing PopC - a story somewhat similar to <a href="http://www.ncbi.nlm.nih.gov/pubmed/11717402">regulation of toxin:antitoxin pairs via antitoxin degradation by Lon protease during nutritional stress</a>.<br />
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All this brings us to the question of importance of the regulated proteolysis during the stringent response. One known example of ppGpp-mediated control via protein degradation is <a href="http://www.ncbi.nlm.nih.gov/pubmed/11474114">degradation of ribosomal proteins by Lon protease induced by accumulation of polyphosphate</a>. Unfortunately, usually stringent response on the whole-cell level is studied <a href="http://www.ncbi.nlm.nih.gov/pubmed/18430135">on the mRNA level, by, say, microarrays</a>. It would be most educational to compare the changes on the mRNA level with changes on the proteome level and to pick up the protein degradation-mediated regulation pathways.<br />
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<b>References:</b><br />
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<span style="font-size: x-small;"><br /></span></div>
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</div>
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Genes+%26+development&rft_id=info%3Apmid%2F9531539&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=The+guanosine+nucleotide+%28p%29ppGpp+initiates+development+and+A-factor+production+in+myxococcus+xanthus.&rft.issn=0890-9369&rft.date=1998&rft.volume=12&rft.issue=7&rft.spage=1022&rft.epage=35&rft.artnum=&rft.au=Harris+BZ&rft.au=Kaiser+D&rft.au=Singer+M&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMolecular+Biology%2C+Developmental+Biology">Harris BZ, Kaiser D, & Singer M (1998). The guanosine nucleotide (p)ppGpp initiates development and A-factor production in <i>Myxococcus xanthus</i>. <span style="font-style: italic;">Genes & Development, 12</span> (7), 1022-35 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/9531539" rev="review">9531539</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Molecular+microbiology&rft_id=info%3Apmid%2F22404381&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=A+RelA-dependent+two-tiered+regulated+proteolysis+cascade+controls+synthesis+of+a+contact-dependent+intercellular+signal+in+Myxococcus+xanthus.&rft.issn=0950-382X&rft.date=2012&rft.volume=&rft.issue=&rft.spage=&rft.epage=&rft.artnum=&rft.au=Konovalova+A&rft.au=L%C3%B6bach+S&rft.au=S%C3%B8gaard-Andersen+L&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CDevelopmental+Biology">Konovalova A, Löbach S, & Søgaard-Andersen L (2012). </span>A RelA-dependent two-tiered regulated proteolysis cascade controls synthesis of a contact-dependent intercellular signal in <i>Myxococcus xanthus</i>. <span style="font-style: italic;">Molecular Microbiology </span> PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/22404381" rev="review">22404381</a></div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-75905245847430339282012-03-13T03:14:00.001-07:002012-03-13T03:14:45.066-07:00Double life of mitochondrial ribosomal protein L7 12<div dir="ltr" style="text-align: left;" trbidi="on">
Mitochondria have their own transcriptional and translational apparatus, even though they produce only a handful of proteins, therefore most of the proteins are imported from the cytoplasm. Trancription, translation and protein insertion into the membrane are interconnected: translational activators regulating mitochondrial translation are interacting with mitochondrial RNA polymerase via Nam1p and Sls1p proteins (<a href="http://www.genetics.org/content/160/1/75.long">Bryan et al. Genetics 2002</a>), Puf proteins connect cytoplasmic translation and protein import into mitochondria by direct interaction with Tom20 subunit of the TOM protein import channel (<a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0002293">Saint-Georges et al. PLoS ONE 2008</a>).<br />
<br />
But this seems not tight enough interaction for mitochondrial translation and transcription. It turnes out what mitohondrial ribosomal protein L7 12 (<a href="http://www.sciencedirect.com/science/article/pii/S0092867405003958">the one that brings translational GTPases to the ribosome</a>), has a double life. Apart from doing its normal job as a part of the ribosome, it doubles as a transctiptional factor, selectively associating with human mitochondrial RNA polymerase and activating it (<a href="http://www.pnas.org/content/108/44/17921.long">Surovtseva et al. PNAS 2011</a>). And as if it is not enough, there are several paralogues of L7 12 in mitochondria, both in plants (<a href="http://www.sciencedirect.com/science/article/pii/S0300908407000193">Delage et al. Biochimie 2007</a>) and in mammals (<a href="http://www.ncbi.nlm.nih.gov/pubmed/11279123">Koc et al. JBC 2001</a>). </div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-60237743754367095902012-03-12T03:59:00.001-07:002012-03-13T02:04:42.697-07:00Measuring nucleotide concentrations inside the living cells<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="float: left; padding-bottom: 5px; padding-left: 5px; padding-right: 5px; padding-top: 5px;"><a href="http://www.researchblogging.org/"><img alt="ResearchBlogging.org" src="http://www.researchblogging.org/public/citation_icons/rb2_large_gray.png" style="border: 0;" /></a></span>
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Taking biological system apart and doing experiments <i>in vitro</i> is a very powerful approach. However, Nature has loads of dirty tricks up her sleeve, so doing experiments <i>in vivo</i> is more kosher - at least you get all the concentrations rights and will have all of the components present in the system.<br />
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<div>
Cells use a whole plethora of nucleotide-based messengers (<a href="http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=19318291&retmode=ref&cmd=prlinks">Pesavento and Hengge, Curr. Opin. Microbiol. 2009</a>), and following concentrations of these <i>in vivo</i> is something microbiologists would love to do. It is possible for some, and c-di-GMP is an example. This nucleotide binds to numerous targets, and one of them is PilZ proteins. When binding to PilZ domain, c-di-GMP promotes a massive structural rearrangement, and this interaction can be monitored by adding a FRET pair to PilZ (<a href="http://www.nature.com/emboj/journal/v26/n24/full/7601918a.html">Benach et al. EMBO J 2007</a>) (<b>Fig. 1</b>). FRET response can be converted in c-di-GMP concentration using a calibration curve, and - viola! - c-di-GMP can be measured in the individual live cells in real time using a PilZ-GFP-based FRET detector (<a href="http://www.sciencemag.org/content/328/5983/1295.short">Christen et al. Science 2010</a>).<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgnZ1qSAWTFduVR0mRKCzNoteyT01tBu9_BushP7OUNEXKhRULzlqQv96Zf9t4TbH3MYiTacaL9nktJrL-TKnOEoaPd4807MzV79AVojYbZFOLqNWB8luzvS4riNVpNTYDl0_H4KEEEYpg/s1600/Screen+Shot+2012-03-12+at+1.34.17+PM.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="226" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgnZ1qSAWTFduVR0mRKCzNoteyT01tBu9_BushP7OUNEXKhRULzlqQv96Zf9t4TbH3MYiTacaL9nktJrL-TKnOEoaPd4807MzV79AVojYbZFOLqNWB8luzvS4riNVpNTYDl0_H4KEEEYpg/s320/Screen+Shot+2012-03-12+at+1.34.17+PM.png" width="320" /></a></div>
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<span style="font-size: x-small;"><b>Fig. 1. </b>PlzD: Apo (A) and in complex with c-di-GMP (figure from <a href="http://www.nature.com/emboj/journal/v26/n24/full/7601918a.html">Benach et al. EMBO J 2007</a>).</span><br />
<br />
The problem with this approach is that is far from being universal. First, one has to have a protein that binds your target nucleotide and undergoes massive rearrangements. Second, this protein should be nice enough to work with so that you can add two <a href="http://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer#CFP-YFP_pairs">GFP molecules to it to make a FRET pair</a>, and still be able to express the protein for <i>in vitro</i> work (one needs to calibrate the FRET response, right?). In the case of stringent response there seem to be no such proteins for detection of my favorite nucleotide, <a href="http://en.wikipedia.org/wiki/Guanosine_pentaphosphate">ppGpp</a>... too bad!</div>
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Well, there seems to be a new method out there, and this one holds great promise. In their recent <a href="http://www.sciencemag.org/content/335/6073/1194.full.pdf">Science paper</a> Paige and colleagues use an RNA-based FRET pair using RNA mimic of GFP (<a href="http://www.sciencemag.org/content/333/6042/642.short">Paige et al. Science 2011</a>) combined with a small-molecule-specific <a href="http://en.wikipedia.org/wiki/Aptamer">aptamer</a> (<b>Fig. 2</b>). When the ligand binds, RNA forms a stable structure and FRET is on! They have followed in <i>E. coli</i> concentrations of two molecules - <a href="http://en.wikipedia.org/wiki/Adenosine_diphosphate">ADP</a> and <a href="http://en.wikipedia.org/wiki/S-Adenosyl_methionine">SAM</a>. However, aptamers can be <a href="http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Retrieve&list_uids=20636061&dopt=abstractplus">evolved for other targets</a>, and this makes this method potentially applicable for detecting whatever molecule that picks your fancy.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiQ-GZ5pyUtpQEOeyhKdjVttOBTP_FaEShUi7hzP7XT-neVxgOmgUmuEBAoF5x8A_Lu3zoGDEFXtlhEpM6CzdfrFS0pCK_53DgPCSV8cFgvYTATJRjosAuxjSqG8MCw7NVuO0nip0Hh1_w/s1600/Screen+Shot+2012-03-12+at+1.11.39+PM.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="104" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiQ-GZ5pyUtpQEOeyhKdjVttOBTP_FaEShUi7hzP7XT-neVxgOmgUmuEBAoF5x8A_Lu3zoGDEFXtlhEpM6CzdfrFS0pCK_53DgPCSV8cFgvYTATJRjosAuxjSqG8MCw7NVuO0nip0Hh1_w/s320/Screen+Shot+2012-03-12+at+1.11.39+PM.png" width="320" /></a></div>
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<span style="font-size: x-small;"><b>Fig. 2. </b>Schematic representartion of the aptamer-based FRET sensor for <i>in vivo</i> detection of small molecules (figure from <a href="http://www.sciencemag.org/content/335/6073/1194.full.pdf">Paige et al. Science 2012</a>).</span></div>
<br />
<b>References:</b><br />
<br /></div>
</div>
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Science+%28New+York%2C+N.Y.%29&rft_id=info%3Apmid%2F22403384&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Fluorescence+imaging+of+cellular+metabolites+with+RNA.&rft.issn=0036-8075&rft.date=2012&rft.volume=335&rft.issue=6073&rft.spage=1194&rft.epage=&rft.artnum=&rft.au=Paige+JS&rft.au=Nguyen-Duc+T&rft.au=Song+W&rft.au=Jaffrey+SR&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CChemistry%2CBiological+Chemistry+%2C+Biophysics%2C+Biotechnology">Paige JS, Nguyen-Duc T, Song W, & Jaffrey SR (2012). Fluorescence imaging of cellular metabolites with RNA. <span style="font-style: italic;">Science (New York, N.Y.), 335</span> (6073) PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/22403384" rev="review">22403384</a></span>
<br />
<br /></div>
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Science+%28New+York%2C+N.Y.%29&rft_id=info%3Apmid%2F21798953&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=RNA+mimics+of+green+fluorescent+protein.&rft.issn=0036-8075&rft.date=2011&rft.volume=333&rft.issue=6042&rft.spage=642&rft.epage=6&rft.artnum=&rft.au=Paige+JS&rft.au=Wu+KY&rft.au=Jaffrey+SR&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CChemistry">Paige JS, Wu KY, & Jaffrey SR (2011). RNA mimics of green fluorescent protein. <span style="font-style: italic;">Science (New York, N.Y.), 333</span> (6042), 642-6 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/21798953" rev="review">21798953</a></span>
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Science+%28New+York%2C+N.Y.%29&rft_id=info%3Apmid%2F20522779&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Asymmetrical+distribution+of+the+second+messenger+c-di-GMP+upon+bacterial+cell+division.&rft.issn=0036-8075&rft.date=2010&rft.volume=328&rft.issue=5983&rft.spage=1295&rft.epage=7&rft.artnum=&rft.au=Christen+M&rft.au=Kulasekara+HD&rft.au=Christen+B&rft.au=Kulasekara+BR&rft.au=Hoffman+LR&rft.au=Miller+SI&rfe_dat=bpr3.included=1;bpr3.tags=Biology">Christen M, Kulasekara HD, Christen B, Kulasekara BR, Hoffman LR, & Miller SI (2010). </span><br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Science+%28New+York%2C+N.Y.%29&rft_id=info%3Apmid%2F20522779&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Asymmetrical+distribution+of+the+second+messenger+c-di-GMP+upon+bacterial+cell+division.&rft.issn=0036-8075&rft.date=2010&rft.volume=328&rft.issue=5983&rft.spage=1295&rft.epage=7&rft.artnum=&rft.au=Christen+M&rft.au=Kulasekara+HD&rft.au=Christen+B&rft.au=Kulasekara+BR&rft.au=Hoffman+LR&rft.au=Miller+SI&rfe_dat=bpr3.included=1;bpr3.tags=Biology"><br /></span><br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Science+%28New+York%2C+N.Y.%29&rft_id=info%3Apmid%2F20522779&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Asymmetrical+distribution+of+the+second+messenger+c-di-GMP+upon+bacterial+cell+division.&rft.issn=0036-8075&rft.date=2010&rft.volume=328&rft.issue=5983&rft.spage=1295&rft.epage=7&rft.artnum=&rft.au=Christen+M&rft.au=Kulasekara+HD&rft.au=Christen+B&rft.au=Kulasekara+BR&rft.au=Hoffman+LR&rft.au=Miller+SI&rfe_dat=bpr3.included=1;bpr3.tags=Biology">Matthias Christen, Hemantha D Kulasekara, Beat Christen, Bridget R Kulasekara, Lucas R Hoffman, and Samuel I Miller (2010) Asymmetrical distribution of the second messenger c-di-GMP upon bacterial cell division. <span style="font-style: italic;">Science (New York, N.Y.), 328</span> (5983), 1295-7 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/20522779" rev="review">20522779</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=The+EMBO+journal&rft_id=info%3Apmid%2F18034161&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=The+structural+basis+of+cyclic+diguanylate+signal+transduction+by+PilZ+domains.&rft.issn=0261-4189&rft.date=2007&rft.volume=26&rft.issue=24&rft.spage=5153&rft.epage=66&rft.artnum=&rft.au=Benach+J&rft.au=Swaminathan+SS&rft.au=Tamayo+R&rft.au=Handelman+SK&rft.au=Folta-Stogniew+E&rft.au=Ramos+JE&rft.au=Forouhar+F&rft.au=Neely+H&rft.au=Seetharaman+J&rft.au=Camilli+A&rft.au=Hunt+JF&rfe_dat=bpr3.included=1;bpr3.tags=Biology">Benach J, Swaminathan SS, Tamayo R, Handelman SK, Folta-Stogniew E, Ramos JE, Forouhar F, Neely H, Seetharaman J, Camilli A, & Hunt JF (2007). The structural basis of cyclic diguanylate signal transduction by PilZ domains. <span style="font-style: italic;">The EMBO journal, 26</span> (24), 5153-66 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/18034161" rev="review">18034161</a></span></div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-90253799423138309722012-01-04T06:50:00.000-08:002012-03-12T07:05:30.830-07:00PhD student in Molecular Evolution with Dr. Gemma Atkinson<div dir="ltr" style="text-align: left;" trbidi="on">
<div dir="ltr" style="text-align: left;" trbidi="on">
<a href="http://lepo.it.da.ut.ee/~atkinson/gem_mac/gemma_c_atkinson.html">Gemma Atkinson</a> is <a href="http://proteinevolution.fieldofscience.com/2012/01/phd-position-available-in-molecular.html">looking for a PhD student in Molecular Evolution</a>.<br />
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The candidate should have:<br />
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- a Masters degree in a biological or computational discipline<br />
- a strong interest in, and enthusiasm for molecular evolution<br />
- familiarity with basic sequence and phylogenetic analyses<br />
- experience in using a programming language such as Python, Perl, Java etc<br />
- fluency in spoken and written English<br />
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The PhD will be funded by a monthly stipend, with additional monies available for regular attendance at international conferences and workshops, and for visiting labs abroad. Information on funding is available by request.<br />
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Applications should contain:<br />
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- a full CV with detailed description of previous relevant experience<br />
- a statement of academic interests<br />
- an electronic version of the Masters thesis<br />
- the names and contact details of at least 2 referees<br />
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The candidate is expected to start at the latest September 2012. Please send applications and informal enquiries to gemma.atkinson@ut.ee<br />
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More information about the research of Dr Atkinson can be found <a href="http://lepo.it.da.ut.ee/~atkinson/gem_mac/gemma_c_atkinson.html">here</a>.
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<b>Update: </b>the call is closed, two applicants selected.</div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-69514500972028817432011-12-14T05:57:00.000-08:002011-12-14T05:57:30.379-08:00Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40<div dir="ltr" style="text-align: left;" trbidi="on">
Most of the mitochondrial proteins are imported from the cytoplasm, with only a small fraction (about 1%) encoded in the mitochondrial genome. Import is mediated by two complexes: <a href="http://en.wikipedia.org/wiki/TIM/TOM_complex">TOM (transporter outer membrane) and TIM (transporter inner membrane)</a>. We have a pretty good idea about the players involved in mitochondrial protein import, but we have very little idea about the dynamics of TOM/TIM movement in the mitochondrial membrane.<br />
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We tried addressing this question using single molecule fluorescent microscopy in isolated yeast mitochondria. What we see is that Tom40, the central component of TOM complex, is highly confined (i.e. restricted in terms of aerea it can sample) but within its confinement it moves pretty rapidly.<br />
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<b>References:</b><br />
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<a href="http://www.nature.com/srep/2011/111214/srep00195/full/srep00195.html">Kuzmenko et al., Scientific Reports (2011) 1:95</a></div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com2tag:blogger.com,1999:blog-2325711757438459748.post-18578495226221963582011-12-02T05:14:00.001-08:002011-12-02T07:40:13.563-08:00long memories of RelA<div dir="ltr" style="text-align: left;" trbidi="on">
Enzymes have their cycles, catalytic ones: bind a substrate, catalyse some sort of reaction, release the product... then do it again. These cycles have memory effects: <a href="http://pubs.rsc.org/en/Content/ArticleLanding/2011/CP/c0cp02860f">long turnover is likely to be followed by another long one, and short one is likely to be followed by another short one</a>. This makes total sense: efficient act of catalysis is possible only when appropriate conformation is achieved and all the residues are aligned as they should be... and that is a recipe for one more efficient round!<br />
<br />
Now let us look at RelA. Based on<a href="http://stringentresponse.blogspot.com/2011/07/single-molecule-investigations-of.html"> <i>in vivo</i> single molecule tracking investigations</a> we recently proposed a model of RelA catalytic cycle: it sits on the ribosome, gets activated by arrival of deacylated tRNA to the A site,<i> falls off</i> and performs <i>multiple</i> acts of ppGpp synthesis from ATP and GDP. Importantly, RelA <i>must</i> go through the ribosome-bound stage in order to get activated. This seems to be an extreme cause of memory effects - while active <i>off</i> the ribosome, RelA remembers the activation event that happened<i> on</i> the ribosome!<br />
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It is quite a scary thought... what else does it remember? Does it remember the moment I started working on it? Well, surely not, even I don't remember that moment any more. Or may be I am just blocking out that memory.<br />
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<b>References:</b><br />
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H. Peter Lu, Phys. Chem. Chem. Phys (2011), 13 pp. 6734-6749, PIMD <a href="http://www.ncbi.nlm.nih.gov/pubmed?term=21409227">21409227</a><br />
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Brian P. English et al., PNAS (2011), 108(13) pp. E365-373, PIMD <a href="http://www.ncbi.nlm.nih.gov/pubmed?term=21730169">21730169</a><br />
<br /></div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-54850498476193282732011-12-02T04:37:00.001-08:002011-12-02T08:14:03.724-08:00Active role of the stringent response in antibiotic tolerance<div dir="ltr" style="text-align: left;" trbidi="on">
From time to time we try killing bacteria with antibiotics. Most of the bugs die, but not all. These survivors fall into two categories: resistant bugs and tolerant bugs. Resistant bugs have specific mechanisms counteracting the drug: mutations in the target site, enzymes destroying the antibiotic, etc. Tolerant bugs are not getting killed using some more general approach, such as forming a biofilm efficiently shielding them from contact with the drug or shutting down its biosynthetic activity and waiting for the better days to come.<br />
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The stringent response is a mechanism rewiring the bacterial physiology under stress. It changes many things simultaneously, and, not surprisingly, <a href="http://onlinelibrary.wiley.com/doi/10.1002/jcp.22158/abstract">functionality of the stringent response is linked to antibiotic tolerance</a>. However, the big question here is the nature of this link: do bugs need functional stringent response in order to tolerate the drug just because relaxed bugs do not shut down their growth when needed and die, or does the stringent response induce production of certain specific enzymes protecting from the drug?</div>
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Recent report by Nguyen and colleagues seems to <a href="http://www.sciencemag.org/content/334/6058/982.short">settle this question</a>. Using series of<i> in vivo </i>experiments with <i>E. coli</i> knock-out strains deficient either in stringent response per se (knock-outs of RelA and SpoT) or in down-stream stringent response-regulated targets they show that the main source of antibiotic tolerance is not a general biosynthetic shut-down. Specifically, they identify two genes induced during the stringent response - <a href="http://en.wikipedia.org/wiki/Superoxide_dismutase">superoxide dismutase</a> (SOD) and <a href="http://en.wikipedia.org/wiki/Catalase">catalase</a> - to be crucial for bacterial survival in the presence of antibacterials. What these do, they protect the bug from the hydroxyl radical. And build-up the latter was recently identified as a <a href="http://www.sciencedirect.com/science/article/pii/S0092867407008999">common mechanism causing the cell death during treatment by different unrelated antibacterials</a>. </div>
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<b>References:</b></div>
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Nguyen at al., Science (2011) 334, pp. 982-986 PIMD <a href="http://www.sciencemag.org/content/334/6058/982.short">22096200</a></div>
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Kohanski et al. Cell (2007) 130, pp. 797-810 PIMD <a href="http://www.sciencedirect.com/science/article/pii/S0092867407008999">17803904</a></div>
</div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-32855302915761947822011-11-03T02:10:00.000-07:002011-11-04T02:16:03.464-07:00DksA and ppGpp regulate transcription of both rRNA and r-proteins<div dir="ltr" style="text-align: left;" trbidi="on">
<div dir="ltr" style="text-align: left;" trbidi="on">
During the stringent response ppGpp together with a small protein DksA bind to the RNA polymerase and down-regulate transcription of the rRNA genes. It makes sense - there is no need for more ribosomes if there are not enough amino acids. However, ribosomes consist not only of rRNA but also of ribosomal proteins, and if the cell stops making rRNA it would make sense to stop making the ribosomal proteins as well.<br />
<br />
And it turnes out that <a href="http://www.pnas.org/content/108/14/5712">ppGpp and DksA do that too</a>. Makes total sense, again. It is quite rare that something about the stringent response makes total sense, but there you are.<br />
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<b>References:</b><br />
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Lemke, J. J., Sanchez-Vazquez, P., Burgos, H. L., Hedberg, G., Ross, W., & Gourse, R. L. (2011). <i>Direct regulation of Escherichia coli ribosomal protein promoters by the transcription factors ppGpp and DksA. </i>Proceedings of the National Academy of Sciences of the United States of America, <b>108</b>(14), 5712–5717.
</div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com0tag:blogger.com,1999:blog-2325711757438459748.post-37088482752197425192011-08-10T05:38:00.000-07:002011-08-14T14:39:34.475-07:00The RelA/SpoT Homolog (RSH) Superfamily: Distribution and Functional Evolution of ppGpp Synthetases and Hydrolases across the Tree of Life<div style="text-align: justify;">Stringent response is run by the RSH (RelA / SpoT Homologue) proteins, but there are more RSHs then just these two. Usually researchers were finding them using an <i>ad hoc</i> approach: take your favorite bug you worked with for 10 years, blast its genome with RelA gene, find anything that looks like RelA, test it.</div><div><div style="text-align: justify;"><br />
</div></div><div><div style="text-align: justify;">Finally there is a proper analysis of RSHs across the tree of life: <a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0023479">The RelA/SpoT Homolog superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life</a> by Atkinson GC, Tenson T, Hauryliuk V, <i>PLoS ONE</i>, 6(<b>8</b>): e23479. </div><div style="text-align: justify;"><br />
</div><div style="text-align: justify;">Here is the take home message:</div><div style="text-align: justify;"></div><ul><li>there are loads of different RSHs out there: we identified 30 subgroups! </li>
<li>all the RSHs out there are now are classified (<i>for now</i>, that is. New genomes are coming out every day, damn the progress!).</li>
<li>Archaea, Bacteria, Eucaryotes: they <i>all</i> have RSHs. I repeat: <b>Archaea</b> too.</li>
<li>there are the long RSHs (i.e. Rel, RelA and SpoT) and there are the short ones. </li>
<li>The short ones have <i>either</i> ppGpp synthesis or ppGpp hydrolysis domain. The long ones have <i>both</i>, but not always both are functional.</li>
<li>by comparing the long ones vs the short ones we identified residues potentially involved in the inter-domain cross-talk in the long ones (the short ones have only <i>one</i> domain thus there is no cross talk there!).</li>
</ul><div>The bottom line: if you work on a strange RSH protein from a strange bug, do check out our paper. </div><div class="separator" style="clear: both; text-align: left;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjE3DH4TWCDhID3R3NZWP9GMyizTa0BccVM8nHzP039NKudgAOqJeBlJtRxTW2Q4eXUsW42Pnvxr9-Zhx-UGrqp09kMyCVsU9ZN4RK5OfIU46UYNCejfcl8c_9VzdQl7E2n5Ar_h0Z7wkg/s1600/FIG1B_HDfasta_FLhit_100_filt_strict_MORECUT_LPR_JUSTDOM_rax_210311.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="640" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjE3DH4TWCDhID3R3NZWP9GMyizTa0BccVM8nHzP039NKudgAOqJeBlJtRxTW2Q4eXUsW42Pnvxr9-Zhx-UGrqp09kMyCVsU9ZN4RK5OfIU46UYNCejfcl8c_9VzdQl7E2n5Ar_h0Z7wkg/s640/FIG1B_HDfasta_FLhit_100_filt_strict_MORECUT_LPR_JUSTDOM_rax_210311.jpg" width="545" /></a></div><b><i>Fig. 1. </i></b> <i>Maximum likelihood phylogeny of the ppGpp hydrolase domain. </i> <i>Subgroups are labeled and shading behind the branches shows the most common domain structure observed for those groups, as per the legend in the inset box. Symbols on branches indicate bootstrap support, as per the inset box.</i><br />
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Hurray to <a href="http://proteinevolution.blogspot.com/">Gem</a>! </div>Stringent Responsehttp://www.blogger.com/profile/10103729906054983242noreply@blogger.com3