Ribosome profiling provides information on the position of ribosomes on mRNA on a genomic scale. Although this information is often used to detect changes in gene expression under different ...conditions, it also has great potential for yielding insight into the mechanism and regulation of protein synthesis itself. First developed in yeast, ribosome profiling involves the isolation and sequencing of ribosome-protected mRNA fragments generated by nuclease treatment. Since the application of ribosome profiling in bacteria has been problematic, we report here a systematically optimized protocol for
that we have used with success for other bacteria as well. Cells are harvested by flash-freezing cultures directly in liquid nitrogen. After lysis, translation is arrested by high magnesium concentration without the use of antibiotics. These improvements eliminate artifacts induced by harvesting cells by centrifugation or filtration and by use of chloramphenicol to arrest translation. These improvements are especially appropriate for studies where the exact position of the ribosome is critical, and not merely the number of ribosomes per message, such as studies aimed at monitoring differences in local elongation rates.
In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ...ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon-anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB's affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.
Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature ...aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells. We find that such RNA damage causes translation stress by stalling elongating ribosomes, which leads to collisions with trailing ribosomes and activation of multiple stress response pathways. Moreover, we discovered a translation-coupled quality control mechanism that resolves covalent RNA-protein crosslinks. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their modification with atypical K6- and K48-linked ubiquitin chains. Ubiquitylation requires the E3 ligase RNF14 and leads to proteasomal degradation of the protein adduct. Our findings identify RNA lesion-induced translational stress as a central component of crosslinking damage.
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.
Getting Past Polyproline Pauses Buskirk, Allen R.; Green, Rachel
Science (American Association for the Advancement of Science),
01/2013, Letnik:
339, Številka:
6115
Journal Article
Recenzirano
Odprti dostop
A protein that associates with the ribosome alleviates stalling and stimulates translation of proline-rich motifs.
Also see Reports by
Ude
et al.
and
Doerfel
et al.
The ribosome is a versatile ...enzyme, but it cannot synthesize all proteins equally well—certain combinations of amino acids pose problems. Ribosome stalling by fairly long amino acid motifs can regulate gene expression in a variety of organisms, from bacteria to humans (
1
,
2
). In these cases, the nascent peptide interacts with the peptide exit channel of the ribosome to induce a conformation that prevents peptide bond formation. Two papers in this issue, by Doerfel
et al.
on page 85 (
3
) and Ude
et al.
on page 82 (
4
), reveal that stalling is common and fundamental: Short, proline (Pro)–rich motifs impede protein synthesis, and stalling is alleviated by a poorly understood elongation factor, EF-P. These studies offer a model of the biological function and mechanism of EF-P through a convergence of biochemical and genetic methods.
Artificial molecular switches that modulate protein activities in response to synthetic small molecules would serve as tools for exerting temporal and dose-dependent control over protein function. ...Self-splicing protein elements (inteins) are attractive starting points for the creation of such switches, because their insertion into a protein blocks the target protein's function until splicing occurs. Natural inteins, however, are not known to be regulated by small molecules. We evolved an intein-based molecular switch that transduces binding of a small molecule into the activation of an arbitrary protein of interest. Simple insertion of a natural ligand-binding domain into a minimal intein destroys splicing activity. To restore activity in a ligand-dependent manner, we linked protein splicing to cell survival or fluorescence in Saccharomyces cerevisiae. Iterated cycles of mutagenesis and selection yielded inteins with strong splicing activities that highly depend on 4-hydroxytamoxifen. Insertion of an evolved intein into four unrelated proteins in living cells revealed that ligand-dependent activation of protein function is general, fairly rapid, dose-dependent, and posttranslational. Our directed-evolution approach therefore evolved small-molecule dependence in a protein and also created a general tool for modulating the function of arbitrary proteins in living cells with a single cell-permeable, synthetic small molecule.
Messenger RNAs lacking a stop codon trap ribosomes at their 3' ends, depleting the pool of ribosomes available for protein synthesis. In bacteria, a remarkable quality control system rescues and ...recycles stalled ribosomes in a process known as trans-translation. Acting as a tRNA, transfer-messenger RNA (tmRNA) is aminoacylated, delivered by EF-Tu to the ribosomal A site, and accepts the nascent polypeptide. Translation then resumes on a reading frame within tmRNA, encoding a short peptide tag that targets the nascent peptide for degradation by proteases. One unsolved issue in trans-translation is how tmRNA and its protein partner SmpB preferentially recognize stalled ribosomes and not actively translating ones. Here, we examine the effect of the length of the 3' extension of mRNA on each step of trans-translation by pre-steady-state kinetic methods and fluorescence polarization binding assays. Unexpectedly, EF-Tu activation and GTP hydrolysis occur rapidly regardless of the length of the mRNA, although the peptidyl transfer to tmRNA decreases as the mRNA 3' extension increases and the tmRNA·SmpB binds less tightly to the ribosome with an mRNA having a long 3' extension. From these results, we conclude that the tmRNA·SmpB complex dissociates during accommodation due to competition between the downstream mRNA and the C-terminal tail for the mRNA channel. Rejection of the tmRNA·SmpB complex during accommodation is reminiscent of the rejection of near-cognate tRNA from the ribosome in canonical translation.
In bacteria, transfer-messenger RNA (tmRNA) and SmpB comprise the most common and effective system for rescuing stalled ribosomes. Ribosomes stall on mRNA transcripts lacking stop codons and are ...rescued as the defective mRNA is swapped for the tmRNA template in a process known as trans-translation. The tmRNA-SmpB complex is recruited to the ribosome independent of a codon-anticodon interaction. Given that the ribosome uses robust discriminatory mechanisms to select against non-cognate tRNAs during canonical decoding, it has been hard to explain how this can happen. Recent structural and biochemical studies show that SmpB licenses tmRNA entry through its interactions with the decoding center and mRNA channel. In particular, the C-terminal tail of SmpB promotes both EFTu activation and accommodation of tmRNA, the former through interactions with 16S rRNA nucleotide G530 and the latter through interactions with the mRNA channel downstream of the A site. Here we present a detailed model of the earliest steps in trans-translation, and in light of these mechanistic considerations, revisit the question of how tmRNA preferentially reacts with stalled, non-translating ribosomes.
N
1
-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m
1
G37 increases ribosomal +1 frameshifting, whether it incurs additional ...translational defects is unknown. Here, we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m
1
G37 deficiency on protein synthesis. Using
E coli
as a model, we show that m
1
G37 deficiency induces ribosome stalling at codons that are normally translated by m
1
G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the affected codons leave the A site. Enzyme- and cell-based assays show that m
1
G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m
1
G37 deficiency similar to those in the stringent response that is typically induced by deficiency of amino acids. This work demonstrates a previously unrecognized function of m
1
G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.
Ribosomes translate genetic information into polypeptides in several basic steps: initiation, elongation, termination and recycling. When ribosomes are arrested during elongation or termination, the ...cell's 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'.
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