Allen R. Buskirk

eIF5A Promotes Translation of Polyproline Motifs

Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis remains elusive. We use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide bond formation between consecutive Pro residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive Pro residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates like Pro.

Nascent peptides that block protein synthesis in bacteria

Significance Ribosomes synthesize all proteins in living cells. There are limits, however, to which sequences they can make. We identified short motifs within translating proteins that inhibit their own synthesis. We developed in vitro methods to determine the molecular mechanism of ribosome stalling by these motifs. Some act by blocking the formation of peptide bonds; in a few of these cases, a translation factor, elongation factor P, alleviates stalling. Other motifs block release of the protein at stop codons. Stalling motifs occur less often than expected in bacterial proteins, suggesting that proteins have evolved to be synthesized efficiently. Although the ribosome is a very general catalyst, it cannot synthesize all protein sequences equally well. For example, ribosomes stall on the secretion monitor (SecM) leader peptide to regulate expression of a downstream gene. Using a genetic selection in Escherichia coli, we identified additional nascent peptide motifs that stall ribosomes. Kinetic studies show that some nascent peptides dramatically inhibit rates of peptide release by release factors. We find that residues upstream of the minimal stalling motif can either enhance or suppress this effect. In other stalling motifs, peptidyl transfer to certain aminoacyl-tRNAs is inhibited. In particular, three consecutive Pro codons pose a challenge for elongating ribosomes. The translation factor elongation factor P, which alleviates pausing at polyproline sequences, has little or no effect on other stalling peptides. The motifs that we identified are underrepresented in bacterial proteomes and show evidence of stalling on endogenous E. coli proteins.

Genetic Identification of Nascent Peptides That Induce Ribosome Stalling

Several nascent peptides stall ribosomes during their own translation in both prokaryotes and eukaryotes. Leader peptides that induce stalling can regulate downstream gene expression. Interestingly, stalling peptides show little sequence similarity and interact with the ribosome through distinct mechanisms. To explore the scope of regulation by stalling peptides and to better understand the mechanism of stalling, we identified and characterized new examples from random libraries. We created a genetic selection that ties the life of Escherichia coli cells to stalling at a specific site. This selection relies on the natural bacterial system that rescues arrested ribosomes. We altered transfer-messenger RNA, a key component of this rescue system, to direct the completion of a necessary protein if and only if stalling occurs. We identified three classes of stalling peptides: C-terminal Pro residues, SecM-like peptides, and the novel stalling sequence FXXYXIWPP. Like the leader peptides SecM and TnaC, the FXXYXIWPP peptide induces stalling efficiently by inhibiting peptidyl transfer. The nascent peptide exit tunnel and peptidyltransferase center are implicated in this stalling event, although mutations in the ribosome affect stalling on SecM and FXXYXIWPP differently. We conclude that ribosome stalling can be caused by numerous sequences and is more common than previously believed.

Clarifying the Translational Pausing Landscape in Bacteria by Ribosome Profiling

The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling.

The role of upstream sequences in selecting the reading frame on tmRNA

BackgroundtmRNA acts first as a tRNA and then as an mRNA to rescue stalled ribosomes in eubacteria. Two unanswered questions about tmRNA function remain: how does tmRNA, lacking an anticodon, bypass the decoding machinery and enter the ribosome? Secondly, how does the ribosome choose the proper codon to resume translation on tmRNA? According to the -1 triplet hypothesis, the answer to both questions lies in the unique properties of the three nucleotides upstream of the first tmRNA codon. These nucleotides assume an A-form conformation that mimics the codon-anticodon interaction, leading to recognition by the decoding center and choice of the reading frame. The -1 triplet hypothesis is important because it is the most credible model in which direct binding and recognition by the ribosome sets the reading frame on tmRNA.ResultsConformational analysis predicts that 18 triplets cannot form the correct structure to function as the -1 triplet of tmRNA. We tested the tmRNA activity of all possible -1 triplet mutants using a genetic assay in Escherichia coli. While many mutants displayed reduced activity, our findings do not match the predictions of this model. Additional mutagenesis identified sequences further upstream that are required for tmRNA function. An immunoblot assay for translation of the tmRNA tag revealed that certain mutations in U85, A86, and the -1 triplet sequence result in improper selection of the first codon and translation in the wrong frame (-1 or +1) in vivo.ConclusionOur findings disprove the -1 triplet hypothesis. The -1 triplet is not required for accommodation of tmRNA into the ribosome, although it plays a minor role in frame selection. Our results strongly disfavor direct ribosomal recognition of the upstream sequence, instead supporting a model in which the binding of a separate ligand to A86 is primarily responsible for frame selection.

SmpB contributes to reading frame selection in the translation of transfer-messenger RNA.

Transfer-messenger RNA (tmRNA) acts first as a tRNA and then as an mRNA template to rescue stalled ribosomes in eubacteria. Together with its protein partner, SmpB (small protein B), tmRNA enters stalled ribosomes and transfers an Ala residue to the growing polypeptide chain. A remarkable step then occurs: the ribosome leaves the stalled mRNA and resumes translation using tmRNA as a template, adding a short peptide tag that destines the aborted protein for destruction. Exactly how the ribosome switches templates, resuming translation on tmRNA in the proper reading frame, remains unknown. Within the tmRNA sequence itself, five nucleotides (U85AGUC) immediately upstream of the first codon appear to direct frame selection. In particular, mutation of the conserved A86 results in severe loss of function both in vitro and in vivo. The A86C mutation causes translation to resume exclusively in the +1 frame. Several candidate binding partners for this upstream sequence have been identified in vitro. Using a genetic selection for tmRNA activity in Escherichia coli, we identified mutations in the SmpB protein that restore the function of A86C tmRNA in vivo. The SmpB mutants increase tagging in the normal reading frame and reduce tagging in the +1 frame. These results demonstrate that SmpB is functionally linked with the sequence upstream of the tmRNA template; both contribute to reading frame selection on tmRNA.

Genetic Analysis of the Structure and Function of Transfer Messenger RNA Pseudoknot 1

tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 106 completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.

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 pausing, arrest and rescue in bacteria and eukaryotes

Ribosomes translate genetic information into polypeptides in several basic steps: initiation, elongation, termination and recycling. When ribosomes are arrested during elongation or termination, the cells capacity for protein synthesis is reduced. There are numerous quality control systems in place to distinguish between paused ribosomes that need some extra input to proceed and terminally stalled ribosomes that need to be rescued. Here, we discuss similarities and differences in the systems for resolution of pauses and rescue of arrested ribosomes in bacteria and eukaryotes, and how ribosome profiling has transformed our ability to decipher these molecular events. This article is part of the themed issue ‘Perspectives on the ribosome’.

A ribosome profiling study of mRNA cleavage by the endonuclease RelE

Implicated in persistence and stress response pathways in bacteria, RelE shuts down protein synthesis by cleaving mRNA within the ribosomal A site. Structural and biochemical studies have shown that RelE cuts with some sequence specificity, which we further characterize here, and that it shows no activity outside the context of the ribosome. We obtained a global view of the effect of RelE on translation by ribosome profiling, observing that ribosomes accumulate on the 5′-end of genes through dynamic cycles of mRNA cleavage, ribosome rescue and initiation. Moreover, the addition of purified RelE to cell lysates shows promise as a method for generating ribosome footprints. In bacteria, profiling studies have suffered from relatively low resolution and have yielded no information on reading frame due to problems inherent to MNase digestion, the method used to degrade unprotected regions of mRNA. In contrast, we find that RelE yields precise 3′-ends that for the first time reveal reading frame in bacteria. Given that RelE has been shown to function in all three domains of life, RelE has potential to improve reading frame and shed light on A-site occupancy in ribosome profiling experiments more broadly.