title: Global analysis of translation termination in E. coli.
abstract: Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America.
authors.forename: Natalie E
authors.initials: NE
authors.lastname: Baggett
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America.
authors.forename: Yan
authors.identifier: 0000-0001-5440-1414
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America.|Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, California, United States of America.|California Institute of Quantitative Biology, University of California, San Francisco, San Francisco, California, United States of America.
authors.forename: Carol A
authors.identifier: 0000-0002-5595-9732
authors.initials: CA
authors.lastname: Gross
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D018388:Codon, Terminator / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D009876:Operon / Q000235:genetics; D010443:Peptide Chain Termination, Translational / Q000235:genetics*; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; C489005:prfA protein, E coli; C489006:prfB protein, E coli; C489007:prfC protein, E coli