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.DOI: http://dx.doi.org/10.7554/eLife.01257.001.
Highlights
To accomplish the huge task of translation elongation—in each cycle, accurately incorporating a new amino acid into a nascent peptide every 1/6th of a second, moving precisely three nucleotides along the messenger RNA (mRNA) template—the ribosome undergoes a series of major structural rearrangements (Figure 1)
Estimating codon-specific occupancy as described in more detail below, we found that the shortage of His-tRNA dramatically increased the relative abundance of large footprints from ribosomes with His codons in the are delivered to the decoding site (A site), with minimal effect on the abundance of small footprints (Figure 4B,C, Figure 4—figure supplement 1)
A ribosome must cycle through a series of consecutive associations with mRNA to decode the message one codon at a time
Summary
To accomplish the huge task of translation elongation—in each cycle, accurately incorporating a new amino acid into a nascent peptide every 1/6th of a second, moving precisely three nucleotides along the mRNA template—the ribosome undergoes a series of major structural rearrangements (Figure 1) (reviewed in Chen et al, 2012 and Noeske and Cate, 2012). The ribosome undergoes a massive rearrangement in which the ribosomal subunits rotate relative to each other (Frank and Agrawal, 2000; Zhang et al, 2009) Along with this rotation, the A and P site tRNAs move from ‘classic’ to ‘hybrid’ states: the anticodon ends stay in their original A and P sites and the acceptor ends move to the P and E sites (Moazed and Noller, 1989; Munro et al, 2007). The A and P site tRNAs move from ‘classic’ to ‘hybrid’ states: the anticodon ends stay in their original A and P sites and the acceptor ends move to the P and E sites (Moazed and Noller, 1989; Munro et al, 2007) This rotated state of the ribosome undergoes additional conformational changes in preparation for translocation (Zhang et al, 2009; Fu et al, 2011). Structural and biochemical studies have revealed many of the atomic-level changes that allow this complicated process to occur (Pulk and Cate, 2013; Tourigny et al, 2013; Zhou et al, 2013), and new details continue to emerge, reshaping models, raising new questions, and leaving other questions still unanswered
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