ppGpp biosyntehsis pathway summed up nicely

A very nice scheme of the ppGpp biosynthesis pathways can be found on the MetaCyc (1). Seems to be a very useful database in general.

References:

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: 22102576

RiboCOURSE Spring 2013: From Ribosome structure to bacterial physiology

An advanced course on bacterial translation and physiology is to be taught at the RiboCORE, Uppsala University in spring 2013. I will be giving a lecture on the stringent response. You can find the course programme here. Review on the stringent response written by Gem Atkinson and myself is a nice introduction to the subject.

GDP and SRL don't mix

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 binding of GDP nucleotide and of SRL rRNA element to translational GTPases IF2 and EF-G are mutually exclusive. 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.

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. 

References:

Mitkevich et al., Scientific Reports 2012 2:843, PIMD: 32150791

First PhD defence in the lab

Our first PhD defence took place on November 2d, 2012. Viktoriya Shyp has defended her work "G nucleotide regulation of translational GTPases and the stringent response factor RelA". Mike Cashel, the discoverer of ppGpp, served as opponent.

Hurray to Vika!

Relacin - a novel antibacterial targeting the stringent response, maybe

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.

A new study is came out in PLoS Pathogens describing a novel Rel inhibitor, relacin (Fig. 1). Wexselblatt and colleagues are following up their earlier work on derivatizing ppGpp into a Rel inhibitor  and are now testing the compound not only in vitro, but also in vivo.

Fig 1: the chemical structure of relacin.

They show that relacin efficiently inhibits sporulation of Bacillus subtilis. Sporulation in this organism is driven by ppGpp, and inhibitory effect of relacin is a strong indication that it actually works. 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. translational GTPases, GTP biosynthesis enzymes etc. The authors do not test these effects. It would be easy to do it in an in vitro translational lysate... but, unfortunately, this is not done.  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.

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.

References:

Wexselblatt et al. Biomed Org Chem (2010) PIMD: 20483622

Wexselblatt et al. PLoS Pathogens (2012) 

ppGpp regulates GTP synthesis by inhibiting Gmk and HprT

Another role for ppGpp was discovered by Allison Kriel and colleagues: it directly regulates GTP levels by interfering with GTP biosynthetic pathway. It is worth saying that inhibitory effects of (p)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 1971, so the connection between (p)ppGpp and metabolism of G nucleotides was known long ago.

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 ppGpp⁰ (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.

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 (Fig. 1). All the experiments are done in B. subtilis, 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.

In  B. subtilis, unlike E. coli, ppGpp does not regulate RNA polymerase directly: the regulation goes via effects on the GTP level. And indeed, a genetic screen performed by Kriel et al. 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 ppGpp⁰ strain - and most of these turned out to be in the GTP biosynthesis pathway. In the E. coli case, the supressor mutations are usually in the RNA polymerase.

 The negative control of GTP synthesis by ppGpp turned out to be crucial for bacterial well-being - in the ppGpp⁰ cells high levels of GTP caused cell death, though the mechanism is still unclear (Fig. 1). Kriel et al. proposed several possible explanations: inhibition with ATP-consuming enzymes, excessive up-regulation of the rRNA transcription, effects on dGTP synthesis etc.

Fig. 1: ppGpp's role in B. subtilis survival via regulation of GTP biosynthesis. Figure from Kriel et al.

References:

Kriel et al, Cell (2012) in press

Gallant et al. JBC (1971), 246 (18): 5812-5816, PIMD: 4938039

Krasny and Grouse, EMBO J. (2004), 23(22): 4473-83, PIMD: 15496987

One more GTPase that binds ppGpp: RF3

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.

Now Kihira and collegues has added one more 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, exchanges GDP to GTP and kicks the class 1 factors off. ppGpp:RF3 complex is not as active as GDP:RF3 one, a situation similar to that with initiation factor 2, IF2. 

The question is is it a specific regulatory mechanism or are GTPases just promiscuous enough? After all, even archael elongation factor 1A, aEF1A, is inhibited by ppGpp as well. And these two never meet in nature...

Kanjee and collegues have recently compiled a list of proteins that ppGpp binds to... or is expected to bind to. GTPases are there, as a whole class of enzymes.


References:

Kihira et al. FEBS J. (2012) in press

Kanjee et al. Mol. Microbiology (2012): 85 (6) 1029-1043 PIMD: 22812515