Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments.
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.DOI: http://dx.doi.org/10.7554/eLife.01257.001.
Department of Biochemistry, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, United States lareau@berkeley.edu.
Liana F
LF
Lareau
Department of Biochemistry, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, United States.
Dustin H
DH
Hite
Department of Biochemistry, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, United States.
Gregory J
GJ
Hogan
Department of Biochemistry, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, United States.
Patrick O
PO
Brown
D059014:High-Throughput Nucleotide Sequencing; D010441:Peptide Chain Elongation, Translational; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger
ribosome; ribosome profiling; translation