Hani S. Zaher

Fidelity at the Molecular Level: Lessons from Protein Synthesis

The faithful and rapid translation of genetic information into peptide sequences is an indispensable property of the ribosome. The mechanistic understanding of strategies used by the ribosome to achieve both speed and fidelity during translation results from nearly a half century of biochemical and structural studies. Emerging from these studies is the common theme that the ribosome uses local as well as remote conformational switches to govern induced-fit mechanisms that ensure accuracy in codon recognition during both tRNA selection and translation termination.

Quality control by the ribosome following peptide bond formation

The overall fidelity of protein synthesis has been thought to rely on the combined accuracy of two basic processes: the aminoacylation of transfer RNAs with their cognate amino acid by the aminoacyl-tRNA synthetases, and the selection of cognate aminoacyl-tRNAs by the ribosome in cooperation with the GTPase elongation factor EF-Tu. These two processes, which together ensure the specific acceptance of a correctly charged cognate tRNA into the aminoacyl (A) site, operate before peptide bond formation. Here we report the identification of an additional mechanism that contributes to high fidelity protein synthesis after peptidyl transfer, using a well-defined in vitro bacterial translation system. In this retrospective quality control step, the incorporation of an amino acid from a non-cognate tRNA into the growing polypeptide chain leads to a general loss of specificity in the A site of the ribosome, and thus to a propagation of errors that results in abortive termination of protein synthesis.

Hyperaccurate and Error-Prone Ribosomes Exploit Distinct Mechanisms during tRNA Selection

Escherichia coli strains displaying hyperaccurate (restrictive) and ribosomal ambiguity (ram) phenotypes have long been associated with alterations in rpsL and rpsD/rpsE, respectively. Crystallographic evidence shows the ribosomal proteins S12 and S4/S5 (corresponding to these genes) to be located in separate regions of the small ribosomal subunit that are important for domain closure thought to take place during tRNA selection. Mechanistically, the process of tRNA selection is separated into two distinct phases, initial selection and proofreading. Here, we explore the effects of mutations in rpsL and rpsD on these steps. Surprisingly, both restrictive and ram ribosomes primarily affect tRNA selection through alteration of the off rates of the near-cognate tRNA species but during distinct phases of the overall process (proofreading and initial selection, respectively). These observations suggest that closure interfaces (S12/h27/h44 versus S4/S5) in two distinct regions of the small ribosomal subunit function independently to promote high-fidelity tRNA selection.

An Active Role for the Ribosome in Determining the Fate of Oxidized mRNA

Chemical damage to RNA affects its functional properties and thus may pose a significant hurdle to the translational apparatus; however, the effects of damaged mRNA on the speed and accuracy of the decoding process and their interplay with quality-control processes are not known. Here, we systematically explore the effects of oxidative damage on the decoding process using a well-defined bacterial in vitro translation system. We find that the oxidative lesion 8-oxoguanosine (8-oxoG) reduces the rate of peptide-bond formation by more than three orders of magnitude independent of its position within the codon. Interestingly, 8-oxoG had little effect on the fidelity of the selection process, suggesting that the modification stalls the translational machinery. Consistent with these findings, 8-oxoG mRNAs were observed to accumulate and associate with polyribosomes in yeast strains in which no-go decay is compromised. Our data provide compelling evidence that mRNA-surveillance mechanisms have evolved to cope with damaged mRNA.

The 2′-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC

The ribosome accelerates the rate of peptidyl transfer by >106‐fold relative to the background rate. A widely accepted model for this rate enhancement invokes entropic effects whereby the ribosome and the 2′‐OH of the peptidyl‐tRNA substrate precisely position the reactive moieties through an extensive network of hydrogen bonds that allows proton movement through them. Some studies, however, have called this model into question because they find the 2′‐OH of the peptidyl‐tRNA to be dispensable for catalysis. Here, we use an in vitro reconstituted translation system to resolve these discrepancies. We find that catalysis is at least 100‐fold slower with the dA76‐substituted peptidyl‐tRNA substrate and that the peptidyl transferase centre undergoes a slow inactivation when the peptidyl‐tRNA lacks the 2′‐OH group. Additionally, the 2′‐OH group was found to be critical for EFTu binding and peptide release. These findings reconcile the conflict in the literature, and support a model where interactions between active site residues and the 2′‐OH of A76 of the peptidyl‐tRNA are pivotal in orienting substrates in this active site for optimal catalysis.

The role of SmpB and the ribosomal decoding center in licensing tmRNA entry into stalled ribosomes

In bacteria, stalled ribosomes are recycled by a hybrid transfer-messenger RNA (tmRNA). Like tRNA, tmRNA is aminoacylated with alanine and is delivered to the ribosome by EF-Tu, where it reacts with the growing polypeptide chain. tmRNA entry into stalled ribosomes poses a challenge to our understanding of ribosome function because it occurs in the absence of a codon-anticodon interaction. Instead, tmRNA entry is licensed by the binding of its protein partner, SmpB, to the ribosomal decoding center. We analyzed a series of SmpB mutants and found that its C-terminal tail is essential for tmRNA accommodation but not for EF-Tu activation. We obtained evidence that the tail likely functions as a helix on the ribosome to promote accommodation and identified key residues in the tail essential for this step. In addition, our mutational analysis points to a role for the conserved K(131)GKK tail residues in trans-translation after peptidyl transfer to tmRNA, presumably EF-G-mediated translocation or translation of the tmRNA template. Surprisingly, analysis of A1492, A1493, and G530 mutants reveals that while these ribosomal nucleotides are essential for normal tRNA selection, they play little to no role in peptidyl transfer to tmRNA. These studies clarify how SmpB interacts with the ribosomal decoding center to license tmRNA entry into stalled ribosomes.

Ribosome-based quality control of mRNA and nascent peptides.

Quality control processes are widespread and play essential roles in detecting defective molecules and removing them in order to maintain organismal fitness. Aberrant messenger RNA (mRNA) molecules, unless properly managed, pose a significant hurdle to cellular proteostasis. Often mRNAs harbor premature stop codons, possess structures that present a block to the translational machinery, or lack stop codons entirely. In eukaryotes, the three cytoplasmic mRNA‐surveillance processes, nonsense‐mediated decay (NMD), no‐go decay (NGD), and nonstop decay (NSD), evolved to cope with these aberrant mRNAs, respectively. Nonstop mRNAs and mRNAs that inhibit translation elongation are especially problematic as they sequester valuable ribosomes from the translating ribosome pool. As a result, in addition to RNA degradation, NSD and NGD are intimately coupled to ribosome rescue in all domains of life. Furthermore, protein products produced from all three classes of defective mRNAs are more likely to malfunction. It is not surprising then that these truncated nascent protein products are subject to degradation. Over the past few years, many studies have begun to document a central role for the ribosome in initiating the RNA and protein quality control processes. The ribosome appears to be responsible for recognizing the target mRNAs as well as for recruiting the factors required to carry out the processes of ribosome rescue and nascent protein decay. WIREs RNA 2017, 8:e1366. doi: 10.1002/wrna.1366

Quality control of chemically damaged RNA.

The “central dogma” of molecular biology describes how information contained in DNA is transformed into RNA and finally into proteins. In order for proteins to maintain their functionality in both the parent cell and subsequent generations, it is essential that the information encoded in DNA and RNA remains unaltered. DNA and RNA are constantly exposed to damaging agents, which can modify nucleic acids and change the information they encode. While much is known about how cells respond to damaged DNA, the importance of protecting RNA has only become appreciated over the past decade. Modification of the nucleobase through oxidation and alkylation has long been known to affect its base-pairing properties during DNA replication. Similarly, recent studies have begun to highlight some of the unwanted consequences of chemical damage on mRNA decoding during translation. Oxidation and alkylation of mRNA appear to have drastic effects on the speed and fidelity of protein synthesis. As some mRNAs can persist for days in certain tissues, it is not surprising that it has recently emerged that mRNA-surveillance and RNA-repair pathways have evolved to clear or correct damaged mRNA.

O6-Methylguanosine leads to position-dependent effects on ribosome speed and fidelity

Nucleic acids are under constant assault from endogenous and environmental agents that alter their physical and chemical properties. O6-methylation of guanosine (m(6)G) is particularly notable for its high mutagenicity, pairing with T, during DNA replication. Yet, while m(6)G accumulates in both DNA and RNA, little is known about its effects on RNA. Here, we investigate the effects of m(6)G on the decoding process, using a reconstituted bacterial translation system. m(6)G at the first and third position of the codon decreases the accuracy of tRNA selection. The ribosome readily incorporates near-cognate aminoacyl-tRNAs (aa-tRNAs) by forming m(6)G-uridine codon-anticodon pairs. Surprisingly, the introduction of m(6)G to the second position of the codon does not promote miscoding, but instead slows the observed rates of peptide-bond formation by >1000-fold for cognate aa-tRNAs without altering the rates for near-cognate aa-tRNAs. These in vitro observations were recapitulated in eukaryotic extracts and HEK293 cells. Interestingly, the analogous modification N6-methyladenosine (m(6)A) at the second position has only a minimal effect on tRNA selection, suggesting that the effects on tRNA selection seen with m(6)G are due to altered geometry of the base pair. Given that the m6G:U base pair is predicted to be nearly indistinguishable from a Watson-Crick base pair, our data suggest that the decoding center of the ribosome is extremely sensitive to changes at the second position. Our data, apart from highlighting the deleterious effects that these adducts pose to cellular fitness, shed new insight into decoding and the process by which the ribosome recognizes codon-anticodon pairs.

Distinct Roles for Release Factor 1 and Release Factor 2 in Translational Quality Control

Background: Bacteria have two similar release factors: release factor 1 and release factor 2 for terminating protein synthesis. Translation terminates prematurely following a misincorporation event. Results: Release factor 2 preferentially recognizes mismatched ribosomal complexes. Conclusion: Release factor 1 and release factor 2 have evolved to carry out distinct functions beyond canonical termination. Significance: Release factor 2 is a key translational quality control factor in bacteria. In bacteria, stop codons are recognized by two similar class 1 release factors, release factor 1 (RF1) and release factor 2 (RF2). Normally, during termination, the class 2 release factor 3 (RF3), a GTPase, functions downstream of peptide release where it accelerates the dissociation of RF1/RF2 prior to ribosome recycling. In addition to their canonical function in termination, both classes of release factor are also involved in a post peptidyl transfer quality control (post PT QC) mechanism where the termination factors recognize mismatched (i.e. error-containing) ribosome complexes and promote premature termination. Here, using a well defined in vitro system, we explored the role of release factors in canonical termination and post PT QC. As reported previously, during canonical termination, RF1 and RF2 recognize stop codons in a similar manner, and RF3 accelerates their rate of dissociation. During post PT QC, only RF2 (and not RF1) effectively binds to mismatched ribosome complexes; and whereas the addition of RF3 to RF2 increased its rate of release on mismatched complexes, the addition of RF3 to RF1 inhibited its rate of release but increased the rate of peptidyl-tRNA dissociation. Our data strongly suggest that RF2, in addition to its primary role in peptide release, functions as the principle factor for post PT QC.