Double life of mitochondrial ribosomal protein L7 12

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 (Bryan et al. Genetics 2002), Puf proteins connect cytoplasmic translation and protein import into mitochondria by direct interaction with Tom20 subunit of the TOM protein import channel (Saint-Georges et al. PLoS ONE 2008).

But this seems not tight enough interaction for mitochondrial translation and transcription. It turnes out what mitohondrial ribosomal protein L7 12 (the one that brings translational GTPases to the ribosome), 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 (Surovtseva et al. PNAS 2011). And as if it is not enough, there are several paralogues of L7 12 in mitochondria, both in plants (Delage et al. Biochimie 2007) and in mammals (Koc et al. JBC 2001). 

DksA and ppGpp regulate transcription of both rRNA and r-proteins

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.

And it turnes out that ppGpp and DksA do that too. Makes total sense, again. It is quite rare that something about the stringent response makes total sense, but there you are.

References:

Lemke, J. J., Sanchez-Vazquez, P., Burgos, H. L., Hedberg, G., Ross, W., & Gourse, R. L. (2011). Direct regulation of Escherichia coli ribosomal protein promoters by the transcription factors ppGpp and DksA. Proceedings of the National Academy of Sciences of the United States of America, 108(14), 5712–5717.

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

Playing chicken, single molecule

Many things happen to DNA. Proteins bind, slide along, dissociate. Sometimes they bump into each other, and then... what happens then?

This was exactly the question adressed in Finkelstein at al., Nature 2010. They were particularly interested in a bacterial protein called RecBCD, which is a powerful helicase. Using single-molecule microscopy they
had a look at what happens when RecBCD rams into some other protein.

And they had several to look at. RNA polymerase was the first one, and this is a formidable roadblock, and yet - RecBCD pushed it off the DNA with ease. Here are the actual images:

RecBCD sliding RNA polymerase along the DNA (76.5 %):

RecBCD ejecting RNA polymerase (8.5 %):

And RecBCD being stalled by RNA polymerase (rare events!) (15 %):

Then they tried other proteins, EcoRI endonuclease and lac repressor, and again, RecBCD was victorious over and over. Only the mighty nucleosome (never mind that it is a eukaryotic protein and it never met RecBCD before!) managed to put up a fight - 24% of the head-on collisions resulted with RecBCD being stalled, 11% resulted in nuceosome ejection and in remaining 65% RecBCD was sliding the nucleosome along the DNA.

What next? Well, now authors are preparing for experiments on RecBCD head-on collisions with Higgs bosons in the LHC.  I bet on RecBCD.

PS: it is all very convenient that the authors happened to study RecBCD - neat story, their protein is stronger than all the others, yay! But wat if they would have started with some lame one, like PNA polumerase? But hey! I never write my results in the order I get them... do you? Did THEY?

References:

Finkelstein IJ, Visnapuu ML, & Greene EC (2010). Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase. Nature, 468 (7326), 983-7 PMID: 21107319

Viral nature of the mitochondrial RNA polymerase

Mitochondria contain their own genome, and they transcribe it. Since mitochondria are of bacterial origin, one would expect that their polymerase would be similar to that of bacteria. And it is so the case for chloroplasts, which are also of bacterial origin.

However, mitochondrial polymerase not homologous to that of bacteria, or, for that matter, to cytosolic eukariotic polymerases. It is homologous to... polymerases of T phages, T3 and T7!

However, it is slightly modified. It has an extension at the N-terminus, and this extension is highly variable. Using this extension yeast mitochondrial polymerase interacts with protein Nam1p, which is involved in mRNA stabilization. Nam1p in turn interacts with the whole bunch of mitochondrial membrane proteins which organise translation and following assembly of the proteins constituting the core of cytochrome c oxidase complex (COX).

In this way all the steps of COX formation are connected in the mitochondria: polimerase binds to Nam1p, Nam1p binds to membrane-bound translational enhancers, enhancers bind mRNA and the ribosome, the ribosome itself binds to membrane, and translated protein is inserted in the membrane. There is even a special term for this sort of coupled transcription, translation and insertion - transertion. Very neat system!

Amazingly, different eukaryotes reinvent this system in different ways. Most of the components are clade-specific, and since the system is so very interconnected, it is is very, very different in different eucaryotes. Therefore when yeast geneticists say that they are studying Saccharomyces cerevisiae as a model system in order to understand human mitochondrial translation they are... well... how should I say it? well, they are being over-optimistic.

PS: and phages and mitochondria are trading polymerases both ways! A cyanophage was discovered that has an ex-mitochondrial DNA (not RNA) polymerase!

References:

Masters BS, Stohl LL, & Clayton DA (1987). Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7. Cell, 51 (1), 89-99 PMID: 3308116

Rodeheffer MS, Boone BE, Bryan AC, & Shadel GS (2001). Nam1p, a protein involved in RNA processing and translation, is coupled to transcription through an interaction with yeast mitochondrial RNA polymerase. The Journal of biological chemistry, 276 (11), 8616-22 PMID: 11118450

Naithani S, Saracco SA, Butler CA, & Fox TD (2003). Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae. Molecular biology of the cell, 14 (1), 324-33 PMID: 12529447

Gagliardi D, Stepien PP, Temperley RJ, Lightowlers RN, & Chrzanowska-Lightowlers ZM (2004). Messenger RNA stability in mitochondria: different means to an end. Trends in genetics : TIG, 20 (6), 260-7 PMID: 15145579

Yi-Wah Chan, Remus Mohr, Andrew D. Millard, Antony B. Holmes, Anthony W. Larkum, Anna L. Whitworth, Nicholas H. Mann, David J. Scanlan, Wolfgang R. Hess and Martha R. J. Clokie. Discovery of cyanophage genomes which contain mitochondrial DNA polymerase. Mol Biol Evol (2011) doi: 10.1093/molbev/msr041