Termination of Protein Synthesis in Mammalian Mitochondria

Cell free synthesis has been readily achieved for the eukaryotic and eubacterial system and wheat germ lysates are also commercially available. Why should mitochondrial systems be so much more difficult? Almost all of the initial attempts to establish in vitro mitochondrial translation systems started with a heterologous system often combining yeast or occasionally mouse mt-mRNAs, or simply homoribopolymers together with a mix of yeast and eubacterial components . Short peptides could be synthesised from poly(U) or poly(UG) templates but only in the presence of eubacterial supplements, whilst programming with either poly(A) or poly(C) templates resulted in two orders of magnitude less product . Other groups reported no nascent peptide production, or in some cases truncated or aggregated products . Explanations proposed for these aborted syntheses included the change of UGA to a tryptophan codon, lack of recognition of the correct AUG initiator, or the necessity for organelle specific tRNAs . The aggregation previously detected could occur either in the exit tunnel or as the peptide emerges from the exit site. It is easy to see how each of these scenarios could cause the ribosome to stall and thus generate only truncated species. Another difficulty is isolating translation competent mammalian mitoribosomes. As mentioned earlier, preparations of isolated mammalian mitoribosomes retain a deacylated P-site tRNA even after puromycin treatment and therefore could not be used. The alternative would be the daunting task of over-expression and purification of the 80+ components and constituents, including presumably modified, mt-rRNAs. This would be especially challenging, as there would be no guarantee that once purified and combined, these components would behave as the eubacterial preparations and dutifully self-assemble. All of the, sadly abortive, attempts so far point towards the need for an in vitro translation system to contain very specific mitochondrial factors from a homologous system. It seems highly probable that a successful system will also require the presence of a membrane into which the products can be co-translationally inserted to prevent aggregation, and the relevant chaperones. It may also prove that critical components have yet to be identified. There is no doubt that having such a system would greatly enhance our progress in understanding post-transcriptional gene expression in mammalian mitochondria. Unfortunately this commodity may remain elusive a while longer. Without it, even the most impressive high resolution structural analyses may only be giving a partial picture.

Termination of protein synthesis in mammalian mitochondria.

Mechanism of Protein Biosynthesis in Mammalian Mitochondria

Protein synthesis in mammalian mitochondria produces 13 ..

Does this additional density derive from 5S rRNA? If so then it seems likely that this density will correspond to only a fragment of the entire 5S, necessitating a form of processing. Is an alternative possibility that this RNA species is only transiently associated with the mt-LSU and is not present in the fully assembled 55S particle? Although plentiful, the copies of cytosolic rRNAs vastly outnumber those of the mt-rRNAs. Moreover due to nuclear encoded but mitochondrially destined proteins that are co-translationally translocated across the outer mitochondrial membrane (OMM), it is difficult to purify mitochondria without cytosolic ribosomes anchored as co-contaminants. Protease shaving and RNase treatment with or without disruption of the OMM can significantly reduce but rarely eliminates all the 18S, 28S and 5S present in preparations, making qPCR analysis unreliable. So how might it be possible to convincingly discriminate between bono fide mitochondrial 5S and a contaminating population? We have performed a simple RNA isolation from fractions following isokinetic sucrose density gradients, which separates the mitoribosomal and cytosolic ribosomal subunits in total cell preparations. If 5S rRNA were present in fully assembled mt-LSU, it would be found in stoichiometric amounts relative to 16S rRNA species. A northern blot following such a fractionation of HEK293 cells is shown in . Probing for major rRNA components (5S, 12S, 16S, 18S and 28S) indicated that a small fraction of the 5S is incorporated into a low density particle as has been well described, previously , whilst the vast majority is associated with the 80S particle as indicated by the co-migration of 18S and 28S rRNA (). Clearly, there is no significant pool of 5S rRNA co-migrating with the16S rRNA, a marker of the mt-LSU, precluding any possibility that the 5S rRNA is present in stoichiometric amounts within the mt-LSU. Further, if the rRNA present is a processed shorter form of 5S, then it avoids detection by standard northern blot using the entire 5S species as probe. It is intriguing that a second RNA species is present in the porcine mt-LSU, but this simple fractionation experiment shows it is unlikely to be the 5S rRNA, unless it is weakly bound and/or subject to degradation.

Organization and Regulation of Mitochondrial Protein Synthesis

Returning briefly to the issue of 5S rRNA import into mammalian mitochondria, this is an issue that has evoked much discussion. Mitochondria from many organisms require RNA species to be imported from the cytosol to support protein synthesis. This is particularly well recognised for transfer RNAs (reviewed in ), where 2 import systems have been characterised . In contrast, PNPase has been implicated in augmenting the import of various other RNA species into human mitochondria, including the 5S rRNA. This surprising observation is supported in part by the main submitochondrial location of PNPase being between the two membranes. Although this protein is better known for its ability to degrade rather than transport RNA , Wang et al. performed a large number of in vitro and in vivo experiments in various species to support the claim, including the use of mutated PNPase to separate import and enzymatic functions. Interested readers are recommended to consult this work .

Holt IJ, Lorimer HE and Jacobs HT (2000) Coupled leading‐ and lagging‐strand synthesis of mammalian mitochondrial DNA. Cell 100: 515–524.
This may result inproteins with different composition of amino acids or it may involve just thelength of 3' UTR.

Factor in Mammalian Mitochondrial Protein Synthesis

As mentioned above, mitoribosomes have acquired a number of new protein components. This means that the MRPs can be divided into two groups, new and old and these are roughly equal in number The old group includes those with clear eubacterial orthologues, evidencing the bacterial origin of the mitochondria, which therefore follow a similar nomenclature (e.g. MRPL1 is the orthologue of RPL1). The second group of ‘new’ mitochondrial specific MRPs (reviewed in ) appears to be evolving more rapidly than cytosolic ribosomal proteins and have adopted functions that suggest they do not merely act as fillers to occupy the space generated by the reduced rRNAs . Acquisition of these novel mitoribosomal proteins appears to be through gene duplication or through the requisition of non-ribosomal proteins that have become targeted to mitochondria, often bearing post-translational modifications (discussed in ). One clear example of such gene duplication in mammals results in the presence of MRPS18A, B and C . The difference in function of these distinct isoforms has not yet been elucidated, but tissue specificity, or the formation of specialised ribosomes dedicated to the translation of subsets of mt-mRNAs, are potential explanations. The acquisition and adaptation of pre-existing proteins is a fascinating phenomenon. A case in point is that of MRPL39, originally termed MRPL5 . A heart specific variant of this protein was identified, which displayed sequence similarity to the N-terminal domain of cytosolic threonyl-tRNA synthetase that had maintained its tRNA binding site . Adaptive evolution presumably dispensed with the mid and C-terminal regions, leaving a mitoribosomal protein with a currently undefined function. Has this substantial increase in the relative amount of protein only evolved to shield the rRNA from damaging reactive oxygen species as speculated by a number of groups, or are there other novel functions still waiting to be disclosed?

Existing protein synthesis inhibitors are primarily used as antibiotics, although some of these antibiotics also have anti-parasitic effects.

chains at the ribosomal site of protein synthesis during ..

Protein synthesis inhibitors are in development for tuberculosis and leishmaniasis. As protein synthesis is a biological process that occurs in all organisms, further exploration of protein biosynthesis machinery as therapeutic targets for neglected tropical diseases is warranted. However, inhibitor selectivity for the machinery of the neglected tropical disease over the machinery of the human host will be essential.

The 5’ UTRs of most mRNAs contain a consensus sequence of5’-CCAGCCAUG-3’ involved in the initiation of protein synthesis.

Also inhibits protein synthesis in mitochondria of mammalian ..

Biogenesis of cytosolic ribosomes requires over 170 proteins and a further 70+ small nucleolar RNAs. In contrast, eubacterial ribosomes, the ancestors of mitochondria, appear to require only 20 or so non-ribosomal proteins to coordinate assembly if one excludes the rRNA modifying enzymes . Although considerably fewer than the trans-acting factors required for 80S assembly the number of factors identified as playing a role is likely to increase . Remarkably this process of ribosome assembly is one that can be recapitulated in vitro (reviewed in ). The pathway of 55S biogenesis is currently very ill defined. Will putting together mammalian mitoribosomes, which are so different in composition to their counterparts, require many or a minimal number of factors to effect assembly? So far we know of only a few assembly factors, including those that chaperone or modify the mt-rRNAs , and those where their specific role is inferred, as their absence causes a disruption in mitoribosome biogenesis . Ribosomal RNA modifications are limited and need further definition (reviewed in ). In some cases, methyltransferases have been implicated, although it has not been possible to demonstrate direct modification in vitro, possibly because the substrate, in the form of a partially assembled mitoribosomal subunit, is not available or that other co-activators may be required . Mitoribosomal protein modification has also been identified. A thorough review by Koc and Koc has shown the overwhelming majority of MRPs to be either acetylated or phosphorylated . However, the extensive proteomics approach did not determine the effects of these modifications on ribosome biogenesis . With the exception of SIRT3 acting to acetylate MRPL10 , the proteins responsible for effecting the modifications, or indeed their functional significance remain largely undefined. Despite our increase in knowledge, many questions still remain. When does the mt-rRNA become incorporated? Are there specific subcomplexes that are initially formed? Are the mt-LSU and mt-SSU assembled on the IMM surface or in the matrix? Are the rRNA modifications a pre-requisite for incorporation into the subunit or do they occur as part of a quality control process? Is there a quality control process that prevents aberrantly assembly subunits from associating to form a 55S particle?