Unknown,Transcriptomics,Genomics,Proteomics

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Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments

ABSTRACT: During translation elongation, the ribosome ratchets along its mRNA template, incorporating each new amino acid and translocating from one codon to the next. The elongation cycle requires dramatic structural rearrangements of the ribosome. We show here that deep sequencing of ribosome-protected mRNA fragments reveals not only the position of each ribosome but also, unexpectedly, its particular stage of the elongation cycle. Sequencing reveals two distinct populations of ribosome footprints, 28-30 nucleotides and 20-22 nucleotides long, representing translating ribosomes in distinct states, differentially stabilized by specific elongation inhibitors. We find that the balance of small and large footprints varies by codon and is correlated with translation speed. The ability to visualize conformational changes in the ribosome during elongation, at single-codon resolution, provides a new way to study the detailed kinetics of translation and a new probe with which to identify the factors that affect each step in the elongation cycle. Ribosome profiling, or sequencing of ribosome-protected mRNA fragments, in yeast. We assay ribosome footprint sizes and positions in three conditions: untreated yeast (3 replicates) and yeast treated with translation inhibitors cycloheximide (2 replicates) and anisomycin (2 biological replicates, one technical replicate). We also treat yeast with 3-aminotriazole to measure the effect of limited histidine tRNAs on ribosome footprint size and distribution (two treatment durations).

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Liana Lareau

PROVIDER: E-GEOD-58321 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments.

Lareau Liana F LF Hite Dustin H DH Hogan Gregory J GJ Brown Patrick O PO

eLife 20140509

During translation elongation, the ribosome ratchets along its mRNA template, incorporating each new amino acid and translocating from one codon to the next. The elongation cycle requires dramatic structural rearrangements of the ribosome. We show here that deep sequencing of ribosome-protected mRNA fragments reveals not only the position of each ribosome but also, unexpectedly, its particular stage of the elongation cycle. Sequencing reveals two distinct populations of ribosome footprints, 28-3 ...[more]

PMID: 24842990

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Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments

Publications

Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments.

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