A cryo-electron microscopic study of ribosome-bound termination factor RF2

Rawat, U.; Zavialov, Andrey V.; Sengupta, Jayati; Valle, Mikel; Grassucci, Robert A.; Linde, J.; Vestergaard, Bente; Ehrenberg, Måns; Frank, Joachim

doi:10.1038/nature01224

Cited by 231 publications

(112 citation statements)

References 25 publications

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“…1), binds the folded RNA with high affinity (8,9), and in Escherichia coli ribosomes, is essential for the RNA to form its native tertiary structure (10). Biochemical evidence and direct observation by cryoelectron microscopy have suggested that the N-terminal domain of L11 (L11-NTD) occupies different positions during the ribosome cycle and variously contacts elongation factor G (EF-G) (11), release factors 1 and 2 (12)(13)(14), and stringent factor (15). E. coli cells lacking L11-NTD (i.e.…”

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confidence: 99%

Interactions of the N-terminal Domain of Ribosomal Protein L11 with Thiostrepton and rRNA

Bausch

Полякова

Draper

2005

Journal of Biological Chemistry

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Ribosomal protein L11 has two domains: the C-terminal domain (L11-C76) binds rRNA, whereas the N-terminal domain (L11-NTD) may variously interact with elongation factor G, the antibiotic thiostrepton, and rRNA. To begin to quantitate these interactions, L11 from Bacillus stearothermophilus has been overexpressed and its properties compared with those of L11-C76 alone in a fluorescence assay for protein-rRNA binding. The assay relies on 2-amino-butyryl-pyrene-uridine incorporated in a 58-nucleotide rRNA fragment, which gives ϳ15-fold enhancement when L11 or L11-C76 is bound.

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confidence: 99%

Interactions of the N-terminal Domain of Ribosomal Protein L11 with Thiostrepton and rRNA

Bausch

Полякова

Draper

2005

Journal of Biological Chemistry

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“…Domain I is important for RF interaction with RF3 3 (13), and when it was removed or swapped between the RFs, this resulted in a general loss of guanine nucleotide exchange on RF3 (29) and in selective effects on the efficiencies of RF1 and RF2; although the efficiency of RF1 remained unaltered when domain I was removed, the efficiency of RF2 decreased considerably (29). These data may suggest that the interaction of domain I with the GTPase-associated center is different for the two factors, in line with recent cryo-EM observations; that is, despite an overall similarity in the trilobed cryo-EM density of RF1 and RF2 on the ribosome, there is an arc-like extra density, seen close to domain I of RF1, which is absent in the cryo-EM structure of RF2 3 (13), and attributed to the NTD of L11. 3 In contrast, domain I of RF2 appears to interact with the CTD of L11 and L11BR 3 (13).…”

Section: Discussionmentioning

confidence: 99%

“…RF1 recognizes the stop codons UAG and UAA, whereas RF2 recognizes the stop codons UGA and UAA in the A site of the ribosome (11). Recent cryo-EM studies with termination complexes containing RF2 (12,13) and RF1 3 show that these two factors acquire very similar overall conformations on the ribosome. Although they bind to the decoding center on the 30 S subunit and reach up to the peptidyltransferase center on the 50 S subunit to induce release of the nascent peptide chain, they interact with L11BR (12,13) and L11 3 with their flexible domain I.…”

mentioning

confidence: 99%

“…Recent cryo-EM studies with termination complexes containing RF2 (12,13) and RF1 3 show that these two factors acquire very similar overall conformations on the ribosome. Although they bind to the decoding center on the 30 S subunit and reach up to the peptidyltransferase center on the 50 S subunit to induce release of the nascent peptide chain, they interact with L11BR (12,13) and L11 3 with their flexible domain I. These observations are in line with previous suggestions, based on biochemical and genetic experiments, that there are interactions between L11 and the release factors (4, 14 -18).…”

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The Role of Ribosomal Protein L11 in Class I Release Factor-mediated Translation Termination and Translational Accuracy

Bouakaz

Murgola

et al. 2006

Journal of Biological Chemistry

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It has been suggested from in vivo and cryoelectron micrographic studies that the large ribosomal subunit protein L11 and its N-terminal domain play an important role in peptide release by, in particular, the class I release factor RF1. In this work, we have studied in vitro the role of L11 in translation termination with ribosomes from a wild type strain (WT-L11), an L11 knocked-out strain (⌬L11), and an L11 N terminus truncated strain (Cter-L11). Our data show 4 -6-fold reductions in termination efficiency (k cat /K m ) of RF1, but not of RF2, on ⌬L11 and Cter-L11 ribosomes compared with wild type. There is, at the same time, no effect of these L11 alterations on the maximal rate of ester bond cleavage by either RF1 or RF2. The rates of dissociation of RF2 but not of RF1 from the ribosome after peptide release are somewhat reduced by the L11 changes irrespective of the presence of RF3, and they cause a 2-fold decrease in the missense error. Our results suggest that the L11 modifications increase nonsense suppression at UAG codons because of the reduced termination efficiency of RF1 and that they decrease nonsense suppression at UGA codons because of a decreased missense error level.L11 is a highly conserved ribosomal 14.8-kDa protein located at the base of the L7/L12 stalk of the ribosome, which is essential for several steps in protein synthesis (1-5). L11 binds to the nucleotides 1051-1108 of Escherichia coli 23 S rRNA, commonly called the L11 binding region (L11BR) 2 (6), which constitutes the GTPase-associated center, an important sector of the bacterial ribosome, where all of the translational GTPases bind and hydrolyze GTP in the course of their action (7). This is also the site of action for the thiazole antibiotics thiostrepton and micrococcin (8 -10). In addition to the GTPases, some other translational factors interact with L11 and the L11BR in functionally important ways. The class I release factors RF1 and RF2 belong to this group. RF1 recognizes the stop codons UAG and UAA, whereas RF2 recognizes the stop codons UGA and UAA in the A site of the ribosome (11). Recent cryo-EM studies with termination complexes containing RF2 (12, 13) and RF1 3 show that these two factors acquire very similar overall conformations on the ribosome. Although they bind to the decoding center on the 30 S subunit and reach up to the peptidyltransferase center on the 50 S subunit to induce release of the nascent peptide chain, they interact with L11BR (12, 13) and L11 3 with their flexible domain I. These observations are in line with previous suggestions, based on biochemical and genetic experiments, that there are interactions between L11 and the release factors (4, 14 -18).The L11 protein consists of two domains, a tightly folded N-terminal domain (NTD), which is loosely connected to the large compact C-terminal domain (CTD). The CTD of L11 is in stable contact with the L11BR RNA, whereas the NTD can change its position and proximity with respect to the rest of the protein (19). Earlier biochemical and genetic studies i...

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“…Based on a cryo-electron microscopy analysis, it is reported that release factor 2 is incorporated in the ribosome by assuming a different shape from that observed in the crystal structure and by changing its interdomain orientations (38,39). A biochemical analysis revealed that the binding mode of ribosome-recycling factor is different from that of tRNA (40).…”

Section: Structure Comparison Of Ef-p With Trna and Ribosome-binding mentioning

confidence: 99%

Crystal structure of elongation factor P from Thermus thermophilus HB8

Hanawa-Suetsugu

Sekine

Sakai

et al. 2004

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

102

105

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Translation elongation factor P (EF-P) stimulates ribosomal peptidyltransferase activity. EF-P is conserved in bacteria and is essential for cell viability. Eukarya and Archaea have an EF-P homologue, eukaryotic initiation factor 5A (eIF-5A). In the present study, we determined the crystal structure of EF-P from Thermus thermophilus HB8 at a 1.65-Å resolution. EF-P consists of three ␤-barrel domains (I, II, and III), whereas eIF-5A has only two domains (N and C domains). Domain I of EF-P is topologically the same as the N domain of eIF-5A. On the other hand, EF-P domains II and III share the same topology as that of the eIF-5A C domain, indicating that domains II and III arose by duplication. Intriguingly, the N-terminal half of domain II and the C-terminal half of domain III of EF-P have sequence homologies to the N-and C-terminal halves, respectively, of the eIF-5A C domain. The three domains of EF-P are arranged in an ''L'' shape, with 65-and 53-Å-long arms at an angle of 95°, which is reminiscent of tRNA. Furthermore, most of the EF-P protein surface is negatively charged. Therefore, EF-P mimics the tRNA shape but uses domain topologies different from those of the known tRNA-mimicry translation factors. Domain I of EF-P has a conserved positive charge at its tip, like the eIF-5A N domain.

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A cryo-electron microscopic study of ribosome-bound termination factor RF2

Cited by 231 publications

References 25 publications

Interactions of the N-terminal Domain of Ribosomal Protein L11 with Thiostrepton and rRNA

Interactions of the N-terminal Domain of Ribosomal Protein L11 with Thiostrepton and rRNA

The Role of Ribosomal Protein L11 in Class I Release Factor-mediated Translation Termination and Translational Accuracy

Crystal structure of elongation factor P from Thermus thermophilus HB8

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