title: Distinct functional classes of ram mutations in 16S rRNA.
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.
authors.forename: Sean P
authors.initials: SP
authors.lastname: McClory
authors.forename: Aishwarya
authors.initials: A
authors.lastname: Devaraj
authors.forename: Kurt
authors.initials: K
authors.lastname: Fredrick
mesh_terms: D000926:Anticodon / Q000235:genetics*; D003062:Codon / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000926:Anticodon; D003062:Codon; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012346:RNA, Transfer, Amino Acyl; C041460:peptide chain termination release factor 2; D006160:Guanosine Triphosphate
keywords: EF-Tu; decoding; ribosome; tRNA; translation