Doing kinetics in vivo and in vitro - what can go wrong?

When you study an enzyme-catalyzed reaction happening in the cell, there are basically 3 things you want to know:

1) how fast?
2) how sensitive to the substrate concentration?
3) how specific?

In terms of the Michaelis-Menten kinetics that would be k_cat, K_M and k_cat/K_M. These all can be measured in vitro, given that you can purify your protein of interest and set up an assay to follow the reaction. But that's in vitro, you say, and how about in vivo? May be everything is different there? What a pleasing thought for a microbiologist!

Here is one neat example.

RelE, a bacterial toxin that binds to the ribosome and cuts mRNA in the A-site, and I already discussed this very protein here and here. For RelE there is an in vitro system available, and k_cat, K_M and k_cat/K_M were measured, x-ray on the ribosome done and we have a pretty good idea about the nature of its catalysis and its specificity (see this post for details). Or do we? May be an in vivo investigation could uncover the real truth here?

Exactly that was performed here: RelE-mediated cleavage products were analysed in living E. coli, mapped on the mRNA sequence and here are the main conclusions:

1) RelE predominantly cuts within the first 100 codons (5') of the mRNA. Direct quote: "It remains unclear how RelE preferentially exerts its effects from the 5′ end of the coding region. Since we did not observe robust cleavage by RelE across the length of the mRNA as seen for HigB, RelE appears to specifically recognize a conformation or component of the translation complex that is unique to initiation or early elongation."

2) no codon specificity observed. None at all.

Wow, that's different. In vitro enzymology suggested that there is a strong codon specificity, and x-ray data were used to explain the bias, and now... it was all just a dream. So how does it work?

First, 5'-prime specificity. In bacteria, translation is co-transcriptional, which means that when mRNA is transcribed, it is translated immediately, before finishing off the transcription. There is even a physical link between the polymease and the ribosome via NusG. And when mRNA is translated, it is cleaved by RelE. Translation and transcription are starting from the 5'-prime... and so is the RelE cleavage, one would guess. Moreover, if you cleave the mRNA once, translation downstream of the cleavage site stops - and the initial cleavage is likely to happen co-transcriptionally at the 5'-prime and render cleavage at the 3'-prime impossible. Is there any need to involve "a conformation or component of the translation complex that is unique to initiation or early elongation"? I think not.

Second, absence of RelE cleavage specificity. Are we looking at RelE-mediated cleavages or at cleavages in general? Obviously, second: there is no way to identify the cause of the cleavage. And in E. coli there are loads of different RNAses involved in the mRNA degradation (here is a wikipedia entry for you, scroll down a bit), so is there a surprise that after RelE selectively cuts mRNA and a swarm of different RNAses processes in further, initial selective signal is lost? I think not.

So what did we learn here? I am not sure. I guess we learned that doing biochemistry in vivo is a tricky business. Doing controls there is hard, and if you get something directly contradicting all previous in vitro data it is worth considering some faul play - E. coli are evil and they use dirty tricks to mislead researchers!

References:

Hurley JM, Cruz JW, Ouyang M, & Woychik NA (2011). Bacterial toxin RelE mediates frequent codon-independent mRNA cleavage from the 5' end of coding regions in vivo. The Journal of biological chemistry PMID: 21324908

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

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

Burmann BM, Schweimer K, Luo X, Wahl MC, Stitt BL, Gottesman ME, & Rösch P (2010). A NusE:NusG complex links transcription and translation. Science (New York, N.Y.), 328 (5977), 501-4 PMID: 20413501

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

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