Tuesday, February 8, 2011
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.
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