RRC ID 53577
Author McClory SP, Devaraj A, Fredrick K.
Title Distinct functional classes of ram mutations in 16S rRNA.
Journal RNA
Abstract During decoding, the ribosome selects the correct (cognate) aminoacyl-tRNA (aa-tRNA) from a large pool of incorrect aa-tRNAs through a two-stage mechanism. In the initial selection stage, aa-tRNA is delivered to the ribosome as part of a ternary complex with elongation factor EF-Tu and GTP. Interactions between codon and anticodon lead to activation of the GTPase domain of EF-Tu and GTP hydrolysis. Then, in the proofreading stage, aa-tRNA is released from EF-Tu and either moves fully into the A/A site (a step termed "accommodation") or dissociates from the ribosome. Cognate codon-anticodon pairing not only stabilizes aa-tRNA at both stages of decoding but also stimulates GTP hydrolysis and accommodation, allowing the process to be both accurate and fast. In previous work, we isolated a number of ribosomal ambiguity (ram) mutations in 16S rRNA, implicating particular regions of the ribosome in the mechanism of decoding. Here, we analyze a representative subset of these mutations with respect to initial selection, proofreading, RF2-dependent termination, and overall miscoding in various contexts. We find that mutations that disrupt inter-subunit bridge B8 increase miscoding in a general way, causing defects in both initial selection and proofreading. Mutations in or near the A site behave differently, increasing miscoding in a codon-anticodon-dependent manner. These latter mutations may create spurious favorable interactions in the A site for certain near-cognate aa-tRNAs, providing an explanation for their context-dependent phenotypes in the cell.
Volume 20(4)
Pages 496-504
Published 2014-4
DOI 10.1261/rna.043331.113
PII rna.043331.113
PMID 24572811
PMC PMC3964911
MeSH Anticodon / genetics* Codon / genetics* Guanosine Triphosphate / metabolism Kinetics Models, Molecular Mutation / genetics* Nucleic Acid Conformation Peptide Termination Factors / chemistry Peptide Termination Factors / genetics Peptide Termination Factors / metabolism Protein Biosynthesis RNA, Messenger / genetics RNA, Ribosomal, 16S / chemistry RNA, Ribosomal, 16S / genetics* RNA, Ribosomal, 16S / metabolism RNA, Transfer, Amino Acyl / chemistry RNA, Transfer, Amino Acyl / genetics* RNA, Transfer, Amino Acyl / metabolism Ribosomes / chemistry Ribosomes / genetics Ribosomes / metabolism*
IF 3.949
Times Cited 11
Prokaryotes E. coli