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

Abstract

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...

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

“…The reduced binding of eEF3 to mutant ribosomes, which favors binding of cognate aminoacyltRNA at the A-site (Uritani and Miyazaki 1988) may also contribute to altered translational fidelity. A similar effect has been previously observed in the case of mutations in E. coli rpS7 (the bacterial rpS5 homolog), which disrupted its interaction with rpS11 (Robert and Brakier-Gingras 2003). The authors likewise suggested that mutations in rpS7 might have impaired the coupling between the E-and the A-site and that this contributed to the reduced translation fidelity (Robert and …”

“…1A; Yusupov et al 2001;Spahn et al 2004) and cross-links to the E-site tRNA (Wower et al 1993;Doring et al 1994). rpS7 also contributes to the formation of the so-called mRNA exit channel and interacts with rpS11, which is located on the platform of the 30S subunit (Robert and Brakier-Gingras 2003). This interaction was suggested to contribute to the structural rearrangements of the head of the 30S subunit during translation (Robert and Brakier-Gingras 2003).…”

“…rpS7 also contributes to the formation of the so-called mRNA exit channel and interacts with rpS11, which is located on the platform of the 30S subunit (Robert and Brakier-Gingras 2003). This interaction was suggested to contribute to the structural rearrangements of the head of the 30S subunit during translation (Robert and Brakier-Gingras 2003). Mutations that disrupt this interaction affect translational fidelity in Escherichia coli, leading to an increased capacity for frameshifting and readthrough (Robert and Brakier-Gingras 2003).…”

“…This interaction was suggested to contribute to the structural rearrangements of the head of the 30S subunit during translation (Robert and Brakier-Gingras 2003). Mutations that disrupt this interaction affect translational fidelity in Escherichia coli, leading to an increased capacity for frameshifting and readthrough (Robert and Brakier-Gingras 2003). In addition, E. coli rpS7 initiates assembly of the 30S subunit by binding to 16S rRNA (Nowotny and Nierhaus 1988;Fredrick et al 2000;Grondek and Culver 2004).…”

“…In Saccharomyces cerevisiae, rpS5 is represented by a single gene copy and is essential for cell viability (Ignatovich et al 1995). The bacterial rpS7/rpS11 interaction is not conserved in eukaryotes (rpS5/rpS14) (Robert and Brakier-Gingras 2003) and the mRNA exit channel formed by the rpS5/ rpS14 interaction is open in isolated yeast 40S subunits (Passmore et al 2007). Interestingly, eukaryotic rpS5 interacts with the cricket paralysis virus (CrPV) IRES (Pfingsten et al 2006;Schuler et al 2006) and was suggested to be one of the determinants that might facilitate recruitment of this IRES to the 40S subunit.…”