Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7

Robert, Françis; Gagnon, Matthieu G.; Sans, Dimitri; Michnick, Stephen W.; Brakier‐Gingras, Léa

doi:10.1017/s1355838200001199

Cited by 11 publications

(11 citation statements)

References 48 publications

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“…However, when the N terminus of S7 is intact but other parts of S7 are deleted, the binding constant decreases but the protein-RNA interaction still takes place. 37 These findings are consistent also with in vivo studies that indicated that when the N terminus of the S7 r-protein is deleted, the assembly efficiency is reduced to about 3% of that observed for full-length S7. 38 Thus, our data reveal a model for a two-stage association of S7 with 16 S rRNA that is supported by other studies in vitro and in vivo, and likely reveal details of bipartite association of r-proteins with 16 S rRNA.…”

Section: Discussionsupporting

confidence: 89%

“…This proposal is consistent with the results of studies in which the binding constants for the complexes formed between 16 S rRNA and fragments of S7 were determined. 37 If the N terminus of S7 is deleted, binding to 16 S rRNA is destroyed. However, when the N terminus of S7 is intact but other parts of S7 are deleted, the binding constant decreases but the protein-RNA interaction still takes place.…”

Section: Discussionmentioning

confidence: 99%

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Temperature-dependent RNP Conformational Rearrangements: Analysis of Binary Complexes of Primary Binding Proteins with 16 S rRNA

Dutcă

Jagannathan

Grondek

et al. 2007

Journal of Molecular Biology

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Ribonucleoprotein particles (RNPs) are important components of all living systems, and the assembly of these particles is an intricate, often multistep, process. The 30 S ribosomal subunit is composed of one large RNA (16 S rRNA) and 21 ribosomal proteins (r-proteins). In vitro studies have revealed that assembly of the 30 S subunit is a temperature-dependent process involving sequential binding of r-proteins and conformational changes of 16 S rRNA. Additionally, a temperature-dependent conformational rearrangement was reported for a complex of primary r-protein S4 and 16 S rRNA. Given these observations, a systematic study of the temperature-dependence of 16 S rRNA architecture in individual complexes with the other five primary binding proteins (S7, S8, S15, S17, and S20) was performed. While all primary binding r-proteins bind 16 S rRNA at low temperature, not all r-proteins/16 S rRNA complexes undergo temperature-dependent conformational rearrangements. Some RNPs achieve the same conformation regardless of temperature, others show minor adjustments in 16 S rRNA conformation upon heating and, finally, others undergo significant temperature-dependent changes. Some of the architectures achieved in these rearrangements are consistent with subsequent downstream assembly events such as assembly of the secondary and tertiary binding r-proteins. The differential interaction of 16 S rRNA with r-proteins illustrates a means for controlling the sequential assembly pathway for complex RNPs and may offer insights into aspects of RNP assembly in general.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Temperature-dependent RNP Conformational Rearrangements: Analysis of Binary Complexes of Primary Binding Proteins with 16 S rRNA

Dutcă

Jagannathan

Grondek

et al. 2007

Journal of Molecular Biology

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“…In this case, protein TthS7 can be regarded as a “natural mutant” of protein EcoS7 [19]. We had previously shown [19, 36] that ThtS7 can form stable complexes with Eco16S. In the present work, the heterologous complex had a K d of 35.8 ± 9.3 nМ ( Fig.…”

Section: Resultsmentioning

confidence: 60%

Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3’-Terminal Fragment of 16S rRNA in E. coli

et al. 2012

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For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNA–protein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNA–protein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UV–induced RNA–protein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA– protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA – protein cross–link within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNA–protein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit.

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“…All the oligonucleotides were from Biocorp Inc. All PCR amplifications were carried out with the Deep Vent DNA polymerase (New England Biolabs) in a Robocycler TM 40 from Stratagene. Plasmid pET21a(ϩ)-S7, which codes for E. coli K12A19 S7 under control of a T7 promoter, and mutant pET-21a(ϩ)-S7⌬148 -178, with a deletion of the last 31 amino acids of S7, are derivatives of pET-21a(ϩ) (Novagen) and were obtained as described (36), except that there was a histidine tag at the C terminus instead of the N terminus of the protein. Mutant pET-21a(ϩ)-S7⌬156 -178 was derived from pET-21a(ϩ)-S7 by introducing a stop codon at position 156, using PCR.…”

Section: Construction Of Plasmids Used In Thismentioning

confidence: 99%

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

Robert¹,

Brakier‐Gingras

2003

Journal of Biological Chemistry

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In this study, we used site-directed mutagenesis to disrupt an interaction that had been detected between ribosomal proteins S7 and S11 in the crystal structure of the bacterial 30 S subunit. This interaction, which is located in the E site, connects the head of the 30 S subunit to the platform and is involved in the formation of the exit channel through which passes the 30 S-bound messenger RNA. Neither mutations in S7 nor mutations in S11 prevented the incorporation of the proteins into the 30 S subunits but they perturbed the function of the ribosome. In vivo assays showed that ribosomes with either mutated S7 or S11 were altered in the control of translational fidelity, having an increased capacity for frameshifting, readthrough of a nonsense codon and codon misreading. Toeprinting and filter-binding assays showed that 30 S subunits with either mutated S7 or S11 have an enhanced capacity to bind mRNA. The effects of the S7 and S11 mutations can be related to an increased flexibility of the head of the 30 S, to an opening of the mRNA exit channel and to a perturbation of the proposed allosteric coupling between the A and E sites. Altogether, our results demonstrate that S7 and S11 interact in a functional manner and support the notion that protein-protein interactions contribute to the dynamics of the ribosome.The ribosome is the cellular machinery responsible for protein synthesis in all living organisms. The elucidation of the crystal structure of the prokaryotic ribosome has led to a major progress in understanding its function (1-7). Analysis of the ribosome structure combined with a wealth of biochemical data clearly showed that ribosomal RNA (rRNA) is the key player in the functions of the ribosome, and it is currently assumed that the main task of the ribosomal proteins is to help and stabilize rRNA folding and to facilitate conformational changes in rRNA (8 -10). However, a growing list of examples suggests that ribosomal proteins directly participate to protein synthesis. The x-ray structure of the ribosome reveals that S12 is part of the decoding site (Refs. 11 and 12, reviewed in Ref. 13). It also shows that the 30 S proteins S13, S15, and the 50S proteins L2, L5, L14, L19 are involved in intersubunit bridges, while the 30 S proteins S9, S13, and the 50 S proteins L1, L5, L33 interact with tRNAs, suggesting that they could participate in the translocation step (12). Studies in solution also point to a role for the ribosomal proteins, such as an involvement in mRNA binding for S1 (14 -16), a participation in peptidyl transferase activity for L2 (17-19) and the prevention of mRNA slippage for L9 (20).Several protein-protein interactions were identified in the crystal structure of the 30 S subunit (21, 22) but, so far, only the interaction between proteins S4 and S5 has been directly related to a particular ribosome function, that is the control of translational fidelity. Indeed, mutations that disrupt the S4-S5 interaction decrease translational accuracy (23,24). Among the other protein-protein inter...

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Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7

Cited by 11 publications

References 48 publications

Temperature-dependent RNP Conformational Rearrangements: Analysis of Binary Complexes of Primary Binding Proteins with 16 S rRNA

Temperature-dependent RNP Conformational Rearrangements: Analysis of Binary Complexes of Primary Binding Proteins with 16 S rRNA

Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3’-Terminal Fragment of 16S rRNA in E. coli

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

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