Site of Functional Interaction of Release Factor 1 with the Ribosome

Dyke, Natalya Van; Murgola, Emanuel J.

doi:10.1016/s0022-2836(03)00537-0

Cited by 22 publications

(24 citation statements)

References 26 publications

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“…In a bacterial strain, containing only the CTD of L11, efficient UAG suppression was seen as in the complete L11 knocked-out strain. This result shows that the NTD of L11 is required for proper functioning of RF1 (18).…”

mentioning

confidence: 79%

“…Bacterial Strains and Plasmids-The three different E. coli strains used for ribosome preparation (6,18) (Table 1) carry a chromosomal knock-out of the rplK gene, which encodes the L11 protein. In the FTP6063 and FTP6066 strains the knock-outs were complemented with the whole L11 gene or its CTD cloned in the p⌬CAT plasmid, which are referred to as pL11 and pL11Cter, respectively (18).…”

Section: Components Of the In Vitro Translation Systemmentioning

confidence: 99%

“…In the FTP6063 and FTP6066 strains the knock-outs were complemented with the whole L11 gene or its CTD cloned in the p⌬CAT plasmid, which are referred to as pL11 and pL11Cter, respectively (18).…”

Section: Components Of the In Vitro Translation Systemmentioning

confidence: 99%

“…We also had functional checks using methods described in Ref. 18. The ⌬L11 ribosomes had minor contaminations of RF1 and RF2 causing release of tetrapeptides during prolonged incubations.…”

Section: Components Of the In Vitro Translation Systemmentioning

confidence: 99%

“…When the recycling of RF1 and RF2 occurred in the presence of an excess amount of RF3 (see "Experimental Procedures"), both factors recycled much more rapidly, but the recycling rates responded to the L11 alterations in a way similar to that in the absence of RF3 (Table 4). Accuracy of mRNA Translation-Studies of termination efficiency in living cells based on nonsense suppression (6,18) are ambiguous in the sense that enhanced nonsense suppression may either be interpreted as reduced termination efficiency of a release factor or enhanced readthrough efficiency of the stop codon by a tRNA. Therefore, we checked whether a complete or an N-terminal domain deletion of the L11 protein affects the accuracy of codon reading (i.e.…”

Section: Termination Of Protein Synthesis By Release Factors In Recycmentioning

confidence: 99%

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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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mentioning

confidence: 79%

Section: Components Of the In Vitro Translation Systemmentioning

confidence: 99%

Section: Components Of the In Vitro Translation Systemmentioning

confidence: 99%

Section: Components Of the In Vitro Translation Systemmentioning

confidence: 99%

Section: Termination Of Protein Synthesis By Release Factors In Recycmentioning

confidence: 99%

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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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Termination and Ribosome Recycling

Wilson

2004

Protein Synthesis and Ribosome Structure

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Domain Reorientation and Induced Fit upon RNA Binding: Solution Structure and Dynamics of Ribosomal Protein L11 from Thermotoga maritima

Il'in¹,

Hoskins²,

Ohlenschläger³

et al. 2005

ChemBioChem

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L11, a protein of the large ribosomal subunit, binds to a highly conserved domain of 23S rRNA and mediates ribosomal GTPase activity. Its C-terminal domain is the main determinant for rRNA binding, whereas its N-terminal domain plays only a limited role in RNA binding. The N-terminal domain is thought to be involved in interactions with elongation and release factors as well as with the antibiotics thiostrepton and micrococcin. This report presents the NMR solution structure of the full-length L11 protein from the thermophilic eubacterium Thermotoga maritima in its free form. The structure is based on a large number of orientational restraints derived from residual dipolar couplings in addition to conventional NOE-based restraints. The solution structure of L11 demonstrates that, in contrast to many other multidomain RNA-binding proteins, the relative orientation of the two domains is well defined. This is shown both by heteronuclear 15N-relaxation and residual dipolar-coupling data. Comparison of this NMR structure with the X-ray structure of RNA-bound L11, reveals that binding not only induces a rigidification of a flexible loop in the C-terminal domain, but also a sizeable reorientation of the N-terminal domain. The domain orientation in free L11 shows limited similarity to that of ribosome-bound L11 in complex with elongation factor, EF-G.

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Site of Functional Interaction of Release Factor 1 with the Ribosome

Cited by 22 publications

References 26 publications

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

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

Termination and Ribosome Recycling

Domain Reorientation and Induced Fit upon RNA Binding: Solution Structure and Dynamics of Ribosomal Protein L11 from Thermotoga maritima

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