Abort! Abort!

Sometimes things go so wrong that it is just easier to start all over again. Bacteria have these situations too - it's not just us, humans! - and the central dogma of molecular biology (DNA replication, transcription and translation) is no exception.

In essence all the three steps of the central dogma share the very same basic topology: there is a message that gets read, there is a tool that reads it and there is a product. It looks like so:

Say, in the case of translation mRNA (the message) gets read by the ribosome (the tool) and protein (the product) is produced. And when things go wrong, there are three things you can abort: the message, the product and the tool. Let us see how it goes.

Replication

DNA polymerase (the tool) reads the DNA (the message) and produces DNA (the product). And when wrong nucleotide is incorporated, DNA polymerase can excise it and continue making the product using so called  proof-reading mechanism. Complete abortion of the growing DNA strand does not happen, and if mistake is done, it is done and you live with it. Surely, there are ways to fix it later (recombination and so on), but not on the spot, during the replication.

Transcription


RNA polymerases can proof-read too. However, many more things can be done. Special set of transcription factors, called GreA and GreB in bacteria and TFSII in eucaryotes, can activate intrinsic hydrolytic activity of the RNA polymerase and cleave off the growing product. Stalled complex is resolved and now we can try again.

Translation


First, there is a proof-reading mechanism, but rather than cutting off the mis-incorporated letter, GTP is hydrolyzed by GTPase EF-Tu which brings the aminoacyl-tRNA.

Second, if the mistake is done, and wrong amino acid was incorporated after all, bacterial class-1 release factors RF1 and RF2 become prone to peptide-release independent of the stop codon, thus removing the product (the growing protein chain). In mitochondria translational system is bacterial-like, but much more insane, and several (as many as 4 in humans!) class-1 release factors are present, with some of them lacking the ability to recognize the stop codon at all (ICT1, for example), and these resolve stalled ribosomal complexes by cutting off the peptide as well as their bacterial counterparts.

Third, bacterial toxins such RelE and the like are resolving ribosomal complexes by cutting the message (mRNA) rather than the product. Calling them toxins is rather misguiding, they are more of the rescue factors.

And lastly, eukaryotic translational factors Dom34 and Hbs1 (related to termination factors eRF1 and eRF3) are splitting the stalled ribosome into subunits, re-setting the tool.

So it seems the further we move from the DNA, the more dispensable the production complex becomes: in the case of DNA polymerases we have only proof-reading, RNA polymerases can do that and also cleave the message, and translational machinery can do it all: cutting the message (RelE), cutting the product (release factors) and resetting the tool by splitting the ribosome into subunits (Dom34 and Hbs).

References:

Borukhov S, Sagitov V, & Goldfarb A (1993). Transcript cleavage factors from E. coli. Cell, 72 (3), 459-66 PMID: 8431948

Toulmé F, Mosrin-Huaman C, Sparkowski J, Das A, Leng M, & Rahmouni AR (2000). GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming. The EMBO journal, 19 (24), 6853-9 PMID: 11118220

Pedersen K, Zavialov AV, Pavlov MY, Elf J, Gerdes K, & Ehrenberg M (2003). The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. Cell, 112 (1), 131-40 PMID: 12526800

Orlova M, Newlands J, Das A, Goldfarb A, & Borukhov S (1995). Intrinsic transcript cleavage activity of RNA polymerase. Proceedings of the National Academy of Sciences of the United States of America, 92 (10), 4596-600 PMID: 7538676

Kassavetis GA, & Geiduschek EP (1993). RNA polymerase marching backward. Science (New York, N.Y.), 259 (5097), 944-5 PMID: 7679800

Richter R, Rorbach J, Pajak A, Smith PM, Wessels HJ, Huynen MA, Smeitink JA, Lightowlers RN, & Chrzanowska-Lightowlers ZM (2010). A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome. The EMBO journal, 29 (6), 1116-25 PMID: 20186120

Shoemaker CJ, Eyler DE, & Green R (2010). Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay. Science (New York, N.Y.), 330 (6002), 369-72 PMID: 20947765

Atkinson GC, Baldauf SL, & Hauryliuk V (2008). Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components. BMC evolutionary biology, 8 PMID: 18947425

Antonicka H, Ostergaard E, Sasarman F, Weraarpachai W, Wibrand F, Pedersen AM, Rodenburg RJ, van der Knaap MS, Smeitink JA, Chrzanowska-Lightowlers ZM, & Shoubridge EA (2010). Mutations in C12orf65 in patients with encephalomyopathy and a mitochondrial translation defect. American journal of human genetics, 87 (1), 115-22 PMID: 20598281

Zaher HS, & Green R (2009). Quality control by the ribosome following peptide bond formation. Nature, 457 (7226), 161-6 PMID: 19092806

Regulation of mitochondrial protein transport

Mitochondria have their own genome, own translational machinery, own ribosomes, but still, most of the proteins they import from the cytosole. And this they do using two protein complexes in the outer and inner membranes: TOM (Transporter Outer Membrane) and TIM (Transporter Inner Membrane). TOM itself consists of several subunits: Tom40 forms a pore through which proteins get transported, Tom20 and Tom70 work as receptors recognizing the mitochondrial proteins in the cytoplasm, and several more proteins helping out.

TOM and TIM, figure lifted from Chacinska at al., 2009

And now joint effort of Pfanner and Meisinger labs lead to a discovery that in yeast TOM-mediated protein transport is regulated by kinases casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes TOM biogenesis, and PKA phosphorylates Tom70 component of TOM under nonrespiring conditions, inhibiting it. This finding basically opens a new field: regulation of mitochondrial protein transport. Just like that.

References:

Chacinska A, Koehler CM, Milenkovic D, Lithgow T, & Pfanner N (2009). Importing mitochondrial proteins: machineries and mechanisms. Cell, 138 (4), 628-44 PMID: 19703392

Schmidt O, Harbauer AB, Rao S, Eyrich B, Zahedi RP, Stojanovski D, Schönfisch B, Guiard B, Sickmann A, Pfanner N, & Meisinger C (2011). Regulation of mitochondrial protein import by cytosolic kinases. Cell, 144 (2), 227-39 PMID: 21215441

Observer effect in biology: Schrödinger's cat mitochondria

All quantum physicists know that observation itself changes the object of observation. We will never know what things are actually doing when we are not looking, just because if in order to figure out what they do, we need to look; it's catch-22. But that's quantum physics, you say. How about molecular biology?

Well, here is an example. Mitochondria, as you know, have their own genome, and they translate it, and they do so in a very funky way. Ever translation termination is peculiar. It is a variation of bacterial translation termination, but different. There are two mitochondrial class-1 release factors (the ones which actually recognize the stop codon and cleave off the peptide): mtRF1a and mtRF1. mtRF1a is an omnipotent release factor and it recognizes normal stop codons UAA and UAG, as it was proved biochemically in vitro. mtRF1... this one is a bit tricky.

First idea is that it recognizes funky stop codons like AGA and AGG (together - AGA/G), which are indeed present in mitochondria. Biochemistry in heterologous system seems to support this one.

Second is that there is no need for mtRF1 at all, and AGA/G stop codons actually never get read at all, therefore there is no need to recognize these! Wow, that's radical and this is why it is published in Science. This story is the subject of this post.

So... how did they figure it out. They check for ribosomal positioning on the termination codon and they figure out that it seems to slip (frame-shift) from the non-standart uAGA/G codon backward and ends up with classical UGAa/g in the A-site. Bang, problem solved, we do not need to recognize the strange stop codon and thus there is no need for mtRF1 at all. Clever. But how do they see it?

They use bacterial toxin RelE. This peculiar molecule binds in the ribosomal A-site and cleaves mRNA there. It works in bacteria, eucaryotes and, obviously, mitochondria because the ribosome is so darn conserved. However, RelE does not cleave all the codons with the same efficiency, it has very strong preferences for certain sequences - such as regular stop codons, UGA or UGG!

Fig. 1 RelE efficiency is different for different codons, lifted from Pedersen at al. 2003

Looking at the x-ray structure of RelE in the complex with mRNA and 70S ribosome we can see why: it is all down to the interactions between the specific residues in RelE and mRNA. If these residues are not there, there will be no interaction and no cleavage - see Fig. 2.

Fig. 2 Proposed reaction mechanism for RelE-mediated cleavage, lifted from Neubauer at al., 2009.

And now - back to the Schrödinger's cat. When researchers used RelE to probe for position of the mitochondrial ribosome on the mRNA, all the cleavages detected were with UAG in the A-site. Why? Well, because this is where RelE can cut, so it cleaved there. It may have even caused this frame-shift. Why didn't they see any ribosomes on the AGG? well, because RelA does not want to cleave there!

So... may be the tool used for observation changed the system and told us something about itself (something that we already knew). Not about the system! Still, it's a Science paper, hey. And the idea is very, very cute!

And, of course, I can be completely wrong!

Fig. 3 Schrödinger's cat. Not really related to RelE at all.


PS: as it turnes out, the problem of affecting the biological system while studying it was discussed by at length here: Bridson EY, & Gould GW, Quantal microbiology.


References:

Neubauer C, Gao YG, Andersen KR, Dunham CM, Kelley AC, Hentschel J, Gerdes K, Ramakrishnan V, & Brodersen DE (2009). The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE. Cell, 139 (6), 1084-95 PMID: 20005802

Andreev D, Hauryliuk V, Terenin I, Dmitriev S, Ehrenberg M, & Shatsky I (2008). The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome. RNA (New York, N.Y.), 14 (2), 233-9 PMID: 18083838

Pedersen K, Zavialov AV, Pavlov MY, Elf J, Gerdes K, & Ehrenberg M (2003). The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. Cell, 112 (1), 131-40 PMID: 12526800

Young DJ, Edgar CD, Murphy J, Fredebohm J, Poole ES, & Tate WP (2010). Bioinformatic, structural, and functional analyses support release factor-like MTRF1 as a protein able to decode nonstandard stop codons beginning with adenine in vertebrate mitochondria. RNA (New York, N.Y.), 16 (6), 1146-55 PMID: 20421313

Soleimanpour-Lichaei HR, Kühl I, Gaisne M, Passos JF, Wydro M, Rorbach J, Temperley R, Bonnefoy N, Tate W, Lightowlers R, & Chrzanowska-Lightowlers Z (2007). mtRF1a is a human mitochondrial translation release factor decoding the major termination codons UAA and UAG. Molecular cell, 27 (5), 745-57 PMID: 17803939

Temperley R, Richter R, Dennerlein S, Lightowlers RN, & Chrzanowska-Lightowlers ZM (2010). Hungry codons promote frameshifting in human mitochondrial ribosomes. Science (New York, N.Y.), 327 (5963) PMID: 20075246

Lekomtsev SA (2007). Non-standard genetic codes and translation termination. Molekuliarnaia biologiia, 41 (6), 964-72 PMID: 18318113

Bridson EY, & Gould GW (2000). Quantal microbiology. Letters in applied microbiology, 30 (2), 95-8 PMID: 10736007

Kinetics of stringent response revealed by microarray analysis

Stringent response is immensely complex and many different proteins are targeted by ppGpp. Different targets have different susceptibility to ppGpp - K_D of binding or K_i of inhibition. Therefore as stringent response progresses and ppGpp concentration changes, different targets will be engaged sequentially, in this way orchestrating changes in the cellular physiology.

Studying the development of stringent response in time is a tricky business. Temporal resolution in one key component, ability following many parameters (many targets) is the other one. Microarrays seem to be the way (Traxler 2006, Traxler 2008, Durfee 2008, Traxler 2011 and Balsalobre 2011).

Here are the main points from Traxler 2011. They did microarray analysis of four strains: wt E. coli K-12-derivative MG1655, ppGpp⁰, Lrp- and RpoS- in MG1655 background. The nature of the wt is important - L12 derivatives are special in that sense that they exhibit stringent response upon L-Valine and L-isolucine overdose, and the latter is used to induce the stringent response in Traxler 2011.

In these strains they follow the effects of two global regulators, Transcription factor Lrp (leucine responsive protein) and RpoS AKA Sigma 38, a specialized σ-factor.

It turnes out that ppGpp regulates both the feed-back loop regulated by Lrp and the feed-forward loop regulated by RpoS (for discussion of how these motifs work see Alon 2007). Moreover, Lrp regulon is induced much earlier than RpoS.

So how is one molecule - ppGpp - evokes two temporally separated responses (early Lrp and late RpoS)? The obvious idea is that these two regulons have different sensitivity to ppGpp, thus the more sensitive one is induced earlier (Lrp) and the less sensitive one is induced later (RpoS). Combining microarray data with measurements of ppGpp concentrations in vivo confirmed this hypothesis.

Rendering Lrp operon inactive in turn disrupts normal progression of the stringent response: both wt and Lpr- strains rapidly accumulate ppGpp, but during the later stage wt cells lower the ppGpp concentration and resume growth, whereas Lrp- strain retains high ppGpp concentration is unable to adapt to stringent conditions. Functional RpoS (which works on the later stages of stringent response) does not help - you have to come prepared, i.e. Lrp regulon should do its job.

However powerful, microarray-based techniques inherently average out the cell-to-cell variability which plays important role in the stringent response as shown by the systems biology investigations.

References:

Matthew F. Traxler, Vineetha M. Zacharia, Stafford Marquard, Sean M. Summers, Huyen-Tran Nguyen, S. Elizabeth Stark and Tyrrell Conway. Discretely calibrated regulatory loops controlled by ppGpp partition gene induction across the ‘feast to famine’ gradient in Escherichia coli. Molecular Microbiology (2011) doi:10.1111/j.1365-2958.2010.07498.x

Matthew F. Traxler, Sean M. Summers, Huyen-Tran Nguyen, Vineetha M. Zacharia, G. Aaron Hightower, Joel T. Smith and Tyrrell Conway. The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli. Molecular Microbiology (2008) 68(5), 1128–1148

Carlos Balsalobre. Concentration matters!! ppGpp, from a whispering to astrident alarmone. Molecular Microbiology (2011) doi:10.1111/j.1365-2958.2010.07521.x

Matthew F Traxler, Dong-Eun Chang, Tyrrell Conway. Guanosine 3',5'-bispyrophosphate coordinates global gene expression during glucose-lactose diauxie in Escherichia coli. PNAS 2006 vol. 103 (7) pp. 2374-9

Tim Durfee, Anne-Marie Hansen, Huijun Zhi, Frederick R. Blattner and Ding Jun Jin. Transcription Profiling of the Stringent Response in Escherichia coli. J. of Bacteriol. 2008, p. 1084-1096, Vol. 190, No. 3

R. I. Leavitt, H. E. Umbarger. Isoleucine and valine metabolism in Escherichia coli. XI. Valine inhibition of the growth of Escherichia coli strain K-12. J. Bacteriol. 1962 vol. 83 pp. 624-30

Uri Alon. Network motifs: theory and experimental approaches. Nat Rev Genet. 2007 vol. 8 (6) pp. 450-61

Brinkman AB, Ettema TJ, de Vos WM, van der Oost J. The Lrp family of transcriptional regulators. Mol Microbiol. 2003 48(2) pp. 287-94

Mendeley group on stringent response

Solving structures with X-ray laser

Single mimivirus particles intercepted and imaged with an X-ray laser and Femtosecond X-ray protein nanocrystallography.

Some details here, for example.

Jet-lagged? it is in your blood

We, animals, have inbuilt metronomes with roughly 24 hour oscillation period, called circadian clocks. These clocks allow organisms to be in sync with the day / night cycle.

And it turnes out that human red blood cells have a circadian clock of their own! And it keeps on ticking when the blood is outside the body!

Peroxiredoxins comprise a conserved family of antioxidant proteins, and researchers checked for peroxiredoxin SO_2/3 oxidation level in human red blood cell samples over a period of time. Amazingly, it oscillated with ∼24 hour period! What makes it particularly interesting is that human red blood cells have no nucleus, therefore no new mRNA can be produced. This means that the cycle is not transcription-driven.

There are several interesting peculiarities of this cycle.

First - it is temperature-sensitive! So... when you have a fever, do your red blood cells speed up their cycle and you get jet-lagged?!

Second - experiments reported in the paper are made using bulk of blood cells rather than on the single cell level. Observed oscillations suggest that cells are somehow synchronized. How? Is it something in the blood? Can we influence this synchronization? Can we re-set the clock?

All this calls for one simple experiment. Take a blood sample in Europe and fly it to the US and check how is it doing there. Is it jet-lagged? Can it adjust?

References:

O'Neill JS, & Reddy AB (2011). Circadian clocks in human red blood cells. Nature, 469 (7331), 498-503 PMID: 21270888

ppGpp mediates cross-talk between the stringent and acid stress responses

We know that stringent response alarmone ppGpp can do about anything, interacting with RNA Polymerase, translational GTPases, Obg GTPase, polynucleotide phosphorylase, DnaG primase, IMP dehydrogenase and adenylosuccinate synthetase to name a few. In net result is: production of ribosomes and tRNAs is halted, cell cycle is arrested, and amino acids are produced.

Well, now one more target was discovered, lysine decarboxylase Ldc1/CadA. Lysine decarboxylase is induced upon acid stress conditions and protects the cell catalyzing decarboxylation of L-lysine resulting in the polyamine cadaverine and carbon dioxide. CO₂ diffuses away and basic cadaverine keeps the intracellular pH, protecting from acidification.

Kanjee at al. solved the Lcd1 x-ray strucutre, and the protein turned out to be in complex with ppGpp. Enzymatic assays showed that ppGpp inhibits Lcd1, thus providing a link between the acid and stringent responses: these two are in a sense are mutually exclusive. During the stringent response ppGpp alters RNA Polymerase in such a way that genes involved in amino acid biosynthesis are predominantly transcribed, and during the acid stress response Lcd1 degrades lysine. Inhibition of Ldl1 by ppGpp stops amino acid degradation, shifting the balance even more towards the amino acid production.

References:

Kanjee U, Gutsche I, Alexopoulos E, Zhao B, El Bakkouri M, Thibault G, Liu K, Ramachandran S, Snider J, Pai EF, & Houry WA (2011). Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase. The EMBO journal PMID: 21278708

Mendeley group on stringent response

photoconvertable GFP variants for SPT

Many different fluorescent labels are used for super-resolution imaging, and photoconvertable GTP variants are probably the most widely used. And again, there are many photoconvertable GFP variants: PATagRFP, PA-GFP, Dendra, EosFP and many others.

What makes a good photoconvertable GFP for single particle tracking (SPT)? Well, all the things that make a good label (bright, stable, fast to mature, small, inert, this convenient and nicely separated adsorbance and emission spectra - the obvious stuff) and one more thing that is not so obvious.

Photoconvertable label should not convert by itself. It should have a stable dark state and conversion should be strictly upon illumination by the photo-converting light. Otherwise creating single molecules for SPT is hard - they just pop into existence by themselves! In than sense EosFP is much better than Dendra.

Sounds obvious, but it wasn't to me...

Increasing signal-to-noise ratio by use of highly inclined thin illumination beam

Signal-to-noise ratio (SNR) is a crucial parameter in imaging. In order to improve it researchers use different tricks, optimizing the labels and reducing the background.

Here is one interesting imaging trick called Highly Inclined and Laminated Optical sheet (HILO) microscopy. Using highly inclined thin imaging beam it is possible to increase SNR dramatically.

In a sense this approach is similar to TIRF, where illuminating light is approaching the sample at a critical angle, so that only molecules very close to the coverslip surface are illuminated by the evanescent wave. The problem with TIRF is exactly that - only molecules very close to the surface can be imaged, which is fine for working with membrane proteins, but not so for the cytosolic ones.

HILO can do just that: instead of illuminating only the molecules in close proximity to the coverslide (as TIRF does) it illuminates a thin slice cutting through the cell at an angle.

References:

Tokunaga et al. Nature Methods 2008, 5 (2) pp. 159-161 PIMD 18176568