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

  • Lareau L
  • Hite D
  • Hogan G
  • et al.
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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.To make a protein from a gene, the gene is first transcribed to produce a molecule of messenger RNA (mRNA), which then passes through a molecular machine called a ribosome. The ribosome reads the genetic code in the mRNA in groups of three letters at a time, and each triplet of letters (or codon) represents an amino acid. The ribosome then joins the relevant amino acids together to build a protein.The ribosome processes about six amino acids per second, on average, but the mRNA is not fed through at a constant rate. Instead, the ribosome changes its shape to ratchet along the mRNA from one codon to the next: it then reads the new codon and adds another amino acid to the protein. However, many of the details of this ratcheting process are not fully understood.In this study, Lareau, Hite et al. have used a technique called ‘ribosome profiling’ to explore the movement of ribosomes along mRNA molecules. First, all of the pieces of mRNA molecules that are not protected inside a ribosome were chemically destroyed. The sequences of the protected fragments were then read and matched to the full-length gene sequences. The protected fragments came in two different sizes: some were about 28–30 letters long, and others were about 20–22 letters long. Lareau, Hite et al. suggest that these different fragment sizes occur because the ribosome switches between two shapes at each codon as it ratchets along the mRNA, and so it protects different lengths of mRNA.In previous ribosome-profiling experiments, the fragments had all been about 28 letters long; but these experiments had used a chemical to halt the progress of the ribosomes along the mRNAs before measuring the length of the fragments. Lareau, Hite et al. argue that this chemical locks the ribosome in the same shape when it brings the ribosome to a halt, and so the protected fragments always have the same length. Further, other chemicals that halt ribosomes appear to lock this molecular machine in the other shape, and so it can only protect the shorter fragments.The findings of Lareau, Hite et al. show that ribosomal profiling experiments can reveal much more than simply where a ribosome is on an mRNA molecule. Further study into the different stages of the ribosome ratcheting process will help uncover how the speed that a ribosome translates an mRNA into a protein can be encoded in the mRNA sequence itself.

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Lareau, L. F., Hite, D. H., Hogan, G. J., & Brown, P. O. (2014). Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments. ELife, 3. https://doi.org/10.7554/elife.01257

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