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R ibosomal am biguity ( ram ) mutations promote 30S domain closure and thereby increase miscoding
Author(s) -
Hoffer Eric D,
Maehigashi Tatsuya,
Fredrick Kurt,
Dunham Christine M.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.628.2
Subject(s) - ribosome , transfer rna , thermus thermophilus , 30s , p site , t arm , biology , genetics , a site , genetic code , ribosomal protein , rna , binding site , dna , gene , escherichia coli
During protein synthesis, ribosomes select correct aminoacyl‐tRNAs by monitoring the nucleotide pairing between the codon on the mRNA and the anticodon of the tRNA in the decoding center. Despite the large pool of near‐cognate tRNAs, the ribosome is highly accurate with an error rate of ~10 −3 – 10 −5 . The process of decoding by the ribosome is thought to be governed by a conformational transition in the ribosome from open to closed that occurs upon codon‐anticodon pairing within the A site. R ibosomal am biguity ( ram ) mutations increase miscoding and map to both ribosomal proteins and rRNA in disparate regions, yet precisely how these mutations act has remained unclear. In this study, we solved crystal structures of Thermus thermophilus 70S ribosomes harboring 16S rRNA ram mutations G299A and G347U in the absence of A‐site tRNA (A‐tRNA) and in the presence of a near‐cognate anticodon stem‐loop. In the absence of an A‐tRNA, each of the mutant ribosomes exhibits a partially closed state. In the 70S‐G347U structure, the 30S shoulder is rotated inward and intersubunit bridge B8 is disrupted. In the 70S‐G299A structure, the 30S shoulder is rotated inward and decoding nucleotide G530 flips into the anti conformation. Both of these mutant ribosomes adopt the fully closed conformation in the presence of near‐cognate A‐tRNA, just as they do with cognate A‐tRNA. Thus, these ram mutations act by promoting the open to closed transition, albeit in somewhat distinct ways. This work reveals the functional importance of 30S shoulder movement for productive aminoacylated‐tRNA incorporation in the A site of the ribosome. Support or Funding Information This work was supported in part by the National Institutes of Health [grant numbers GM093278 to CMD, GM072528 to KF]. The X‐ray crystallography datasets were collected at the at the NE‐CAT beamlines (funded by the NIGMS from the NIH (P30 GM124165)), using a Pilatus detector (RR029205) and an Eiger detector (OD021527), and at the SER‐CAT beamlines (funded by its member institutions, and NIH equipment grants RR25528 and RR028976). This research used resources of the APS, a US Department of Energy Office of Science User Facility operated by Argonne National Laboratory under Contract DE‐AC02‐06CH11357 (NE‐CAT) and W‐31‐109‐Eng‐38 (SER‐CAT). Dr. Dunham is a Burroughs Wellcome Investigator in the Pathogenesis of Infectious Diseases. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .