Abstract
Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a pre- steady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Codon, Terminator / genetics
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Codon, Terminator / metabolism
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Escherichia coli Proteins / chemistry
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Escherichia coli Proteins / metabolism
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Kinetics
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Models, Molecular
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Mutation
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Nucleic Acid Conformation*
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Peptide Termination Factors / chemistry
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Peptide Termination Factors / metabolism
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Protein Biosynthesis
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Protein Conformation
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RNA, Bacterial / chemistry
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RNA, Bacterial / genetics
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RNA, Bacterial / metabolism
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RNA, Ribosomal, 23S / chemistry*
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RNA, Ribosomal, 23S / genetics
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RNA, Ribosomal, 23S / metabolism*
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RNA, Transfer / genetics
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RNA, Transfer / metabolism
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Ribosomes / chemistry
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Ribosomes / genetics
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Ribosomes / metabolism*
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Substrate Specificity
Substances
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Codon, Terminator
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Escherichia coli Proteins
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Peptide Termination Factors
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RNA, Bacterial
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RNA, Ribosomal, 23S
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prfB protein, E coli
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RNA, Transfer