Dom34 Rescues Ribosomes in 3′ Untranslated Regions

Guydosh, Nicholas R.; Green, Rachel

doi:10.1016/j.cell.2014.02.006

Cited by 354 publications

(511 citation statements)

References 68 publications

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“…These results suggest that the stalled ribosome at the 5th codon position might adopt a more compact conformation than the others. Supporting this notion, recent studies demonstrated that the length of footprints could be influenced by ribosome dynamics [20,21].…”

Section: Ribosome Profiling Reveals Post-initiation Pausingmentioning

confidence: 65%

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

et al. 2014

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The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.

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Section: Ribosome Profiling Reveals Post-initiation Pausingmentioning

confidence: 65%

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

et al. 2014

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“…Recently, it was shown that after a termination event, 40S subunits, and probably also 80S ribosomes, could scan along the mRNA downstream or upstream to the next AUG position (64). Ribosome profiling methodology also revealed the possibility that 80S ribosomes scan downstream after translating a cistron in yeast (65). These observations suggest that the scanning mode of 70S or 80S ribosomes might be a universal ribosomal feature of the ribosomal translation process.…”

Section: Discussionmentioning

confidence: 86%

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Yamamoto

Wittek

Gupta

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.protein synthesis | ribosomal functions | translational initiation | 30S-binding initiation | 70S-scanning initiation I t is textbook knowledge that 30S subunits initiate protein synthesis in bacteria; they recognize the initiation site of the mRNA composed of the Shine-Dalgarno (SD) sequence, the AUG codon, and fMet-tRNA, together with three initiation factors (IFs) forming the 30S initiation complex (30SIC). Association of the large 50S subunit triggers the release of the IFs, leading to the 70S initiation complex (70SIC) that enters the elongation phase of translation (reviewed in 1). We term this initiation path the "30S-binding mode" of bacterial initiation. After elongation and termination, it is thought that the ribosome dissociates into its subunits, thus providing 30S subunits for the next round of initiation.The functional role of IF2 is well defined. It can bind directly to the 30S, providing a docking site for fMet-tRNA (2), but it can also enter the 30S subunit as ternary complex fMet-tRNA•IF2•GTP (3). Both IF2 and IF3 are essential for viability. IF3 has a binding site at the 30S interface (4), which explains its antiassociation effect (5, 6), as well as its role in dissociation of the terminating 70S ribosome (7). However, the in vivo concentration of IF3 is 100-fold less (8) than required for full dissociation of 70S in vitro (4). Evidence for the presence of IF3 on 70S ribosomes was reported (9), indicating that the functional spectrum of IF3 is possibly not restricted to an antiassociation effect. Both IF3 and IF2 are also responsible for the fidelity of decoding the initiation AUG by fMet-tRNA Met f at the P site of 30S subun...

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“…Experiments without CHX, on the other hand, report positive correlations of varying magnitude (Fig 1D, 0x Gerashchenko NAR points and purple labels). Experiments by Pop [36], Lareau [26], Nedialkova [29], Guydosh [35] and Gardin [33] produce weak to moderate correlations, but experiments by Gerashchenko [34], Jan [41], Williams [42], Weinberg [38], and Young [37] produce fairly strong and highly statistically significant correlations.…”

Section: Pretreatment With Cycloheximide Consistently Changes Enrichmmentioning

confidence: 99%

“…An alternative experimental protocol uses an optimized harvesting and flash-freezing process that allows CHX pretreatment to be omitted [15,17,26,29,[33][34][35][36][37][38]. Experiments performed with this protocol have revealed that treatment with CHX affects several high-level characteristics of footprinting data, including the distribution of lengths of nuclease-protected fragments in mammalian cells [39] and the amount of enrichment in ribosome density at the 5' end of coding sequences in yeast [34].…”

Section: Introductionmentioning

confidence: 99%

Understanding biases in ribosome profiling experiments reveals signatures of translation dynamics in yeast

Hussmann

Patchett

Johnson

et al. 2015

Preprint

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Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics. Author SummaryRibosome profiling measures the precise locations of millions of actively translating ribosomes on mRNAs. In theory, the frequency with which ribosomes are observed positioned over each type of codon can be used to quantify the speed with which each codon is translated. In practice, ribosome profiling experiments in yeast that use translation inhibitors to arrest translation before measuring the positions of ribosomes report very different Data Availability Statement: Sequencing data has been deposited to the SRA with accession number SRP060588.Funding: This work was supported by NIH grant R01-GM-093086 to SS and NIH grant GM053655 to AJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.apparent translation speeds for each codon than experiments that do not use inhibitors.To explain this inconsistency, we show that a previously unappreciated mechanism causes experiments using translation inhibitors to not measure ribosomes at each position on mRNAs in proportion to the actual amount of time spent there in vivo. Understanding this mechanism reveals that experiments without inhibitors more accurately measure translation dynamics and provides guidance for the design and interpretation of future ribosome profiling experiments.

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Dom34 Rescues Ribosomes in 3′ Untranslated Regions

Cited by 354 publications

References 68 publications

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Understanding biases in ribosome profiling experiments reveals signatures of translation dynamics in yeast

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