Structural basis for the inhibition of the eukaryotic ribosome

Loubresse, Nicolas Garreau de; Prokhorova, I.; Holtkamp, Wolf; Rodnina, Marina V.; Yusupov, Marat

doi:10.1038/nature13737

Cited by 451 publications

(471 citation statements)

References 76 publications

(51 reference statements)

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“…Furthermore, reconstitution of translation in the presence of the elongation inhibitor cycloheximide results in relative movement of the ribosome on the dicistrovirus IRES by six nucleotides, indicating that two cycles of elongation have occurred (6,11,18,19). Cycloheximide inhibits translation by binding to the ribosomal E site, and as such, these observations are consistent with a model in which the IRES initially occupies the P site to direct translation from the A site (18,32). In light of the recent highresolution structural data presenting evidence that initial ribosome binding positions the PKI domain of the IRES in the ribosomal A site, we reevaluated the initial translocation steps of ribosomes assembled on the IRES (20,22).…”

Section: A6554 and A6576 Contribute To Ires-mediated Translation Andsupporting

confidence: 74%

“…5, lane 11). Whereas previous interpretations of the biochemical data indicated that cycloheximide inhibits translation when a deacylated tRNA is bound in the E site (18), given the recent structural data indicating that cycloheximide binds to the E site, where the acceptor end of a tRNA normally resides (32), an alternate interpretation is that cycloheximide induces a premature block in the initial steps of IRES translation by binding to the ribosome and inhibiting translocation with the IAPV PKI domain in the E site and the first aminoacyl-tRNA in the P site. Thus, occupancy of the IAPV tRNAlike PKI domain in the E site can still accommodate cycloheximide binding, leading to inhibition of ribosome translocation.…”

Section: A6554 and A6576 Contribute To Ires-mediated Translation Andmentioning

confidence: 96%

See 1 more Smart Citation

Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

Cornilescu

Mouzakis

et al. 2015

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

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The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the threehelical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.translation | virus | RNA | ribosome | internal ribosome entry site F idelity of protein synthesis and the transmission of genetic information from mRNA into a nascent protein rely on the accurate selection and maintenance of the translational reading frame. In canonical eukaryotic translation, after recruitment and scanning of ribosomes on an mRNA, the translational reading frame is initially established by methionyl-tRNA i anticodon: codon pairing in the ribosomal P site. Although the mechanisms that specify reading frame selection and maintenance during translation are not completely understood, programmed recoding mechanisms that have been identified in some viral and cellular mRNAs have yielded significant insights into the cis-acting signals that increase coding capacity or allow translation using alternate reading frames (1, 2).We recently demonstrated that a subset of viruses within the Dicistroviridae family harbors an intergenic region internal ribosome entry site (IGR IRES) that can direct translation in alternative reading frames (3), providing an excellent model for studying RNA-ribosome interactions that influence reading frame selection. An IRES is generally a structured RNA element that can recruit the ribosome in a 5′ end-independent manner and without the full complement of canonical translation initiation factors (4, 5). Among the diverse types of IRES elements found in both viral and messenger RNAs, the IGR IRES uses the most streamlined mechanism, dispensing the need for all canonical initiation factors to directly recruit the ribosome and initiate translation at a non-AUG codon (6-8). The IGR IRES adopts an RNA structure comprising two independently folded domains; ps...

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Section: A6554 and A6576 Contribute To Ires-mediated Translation Andsupporting

confidence: 74%

Section: A6554 and A6576 Contribute To Ires-mediated Translation Andmentioning

confidence: 96%

Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

Cornilescu

Mouzakis

et al. 2015

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

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“…We have found that DuL11AB cells are not hypersensitive toward anisomycin, a specific inhibitor of peptide bond formation. 46,47 This suggests that the peptidyltransferase reaction is not affected by the lack of the uL11 protein. Complementary, none of the perturbations within the GAC was shown to exert an effect on the peptidyl transferase Figure 5.…”

Section: Ul11 Involvement In Ribosomal Speed and Accuracymentioning

confidence: 98%

Functional analysis of the uL11 protein impact on translational machinery

Wawiórka¹,

Molestak²,

Szajwaj³

et al. 2016

Cell Cycle

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The ribosomal GTPase associated center constitutes the ribosomal area, which is the landing platform for translational GTPases and stimulates their hydrolytic activity. The ribosomal stalk represents a landmark structure in this center, and in eukaryotes is composed of uL11, uL10 and P1/P2 proteins. The modus operandi of the uL11 protein has not been exhaustively studied in vivo neither in prokaryotic nor in eukaryotic cells. Using a yeast model, we have brought functional insight into the translational apparatus deprived of uL11, filling the gap between structural and biochemical studies. We show that the uL11 is an important element in various aspects of 'ribosomal life'. uL11 is involved in 'birth' (biogenesis and initiation), by taking part in Tif6 release and contributing to ribosomal subunit-joining at the initiation step of translation. uL11 is particularly engaged in the 'active life' of the ribosome, in elongation, being responsible for the interplay with eEF1A and fidelity of translation and contributing to a lesser extent to eEF2-dependent translocation. Our results define the uL11 protein as a critical GAC element universally involved in trGTPase 'productive state' stabilization, being primarily a part of the ribosomal element allosterically contributing to the fidelity of the decoding event.

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“…CHX is a small-molecule translation inhibitor that has been a staple of experimental approaches to the study of translation for decades. The exact mechanism of this inhibition, however, is not completely understood, with recent studies suggesting that CHX binds to a ribosome's E-site along with a deacylated tRNA to block further translocation [19,20]. The majority of the rapidly growing body of ribosome profiling experiments in yeast have followed this original CHX-pretreatment protocol [13,[21][22][23][24][25][26][27][28][29][30].…”

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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Structural basis for the inhibition of the eukaryotic ribosome

Cited by 451 publications

References 76 publications

Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

Functional analysis of the uL11 protein impact on translational machinery

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

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