An Investigation into the Effects of Alterations in Ribosomal Proteins S4 and S5 on Translational Accuracy, Ribosomal RNA Processing, and Ribosome Assembly

Abstract

Accurate translation of genetic message is an essential step in gene expression and is carried out by the ribosome. Ribosomal proteins S4 and S5 are critical for the decoding and the assembly processes on the ribosome. The interaction between residues in the S4-S5 interface region is crucial for the large scale conformational changes occurring on the ribosome during decoding. Earlier studies predominantly isolated S4 and S5 mutations located near the S4-S5 interface. However, recent studies show that ribosomal proteins S4 and S5 affect translational accuracy to a larger extent than previously realized. To understand the diverse ways by which mutation in S4 and S5 can affect translational accuracy, we generated and studied a wide array of S4 and S5 mutants with altered accuracy phenotypes, that were located not only at the interface but also distributed throughout the protein. We also generated a collection of mutants with alteration in the loop 2 region of S5 protein. As the loop 2 region is away from the interface, it modulates accuracy by mechanisms different than disruption of the S4-S5 interface. The C-terminal region of S4 protein interacts extensively with the S5 protein and C-terminal truncated mutants are temperature sensitive, have a ribosome assembly defect and are error-prone. We obtained temperature insensitive S4 mutants with restored C-termini that partially rescued miscoding but almost completely rescued the ribosome assembly defect at 42˚C. Finally, in the process of exploring interactions of S4 and S5 with other regions in the ribosome, we discovered that accuracy can be altered indirectly by affecting amino acid pools or tRNA modification.