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Molecular Mechanisms of Translation and Their Implications for Gene Regulation

Research Summary

Rachel Green uses biochemical and whole genome approaches to study the mechanism of translation by the ribosome, and its regulation, in bacterial and eukaryotic systems.

Translation of the genetic information in the cell into functional proteins is an essential and highly conserved function. The ribosome, a two-subunit macromolecular complex, composed in bacteria of three large RNAs and more than 50 proteins, is the catalyst and framework for the precise and coordinated process of translation. The small subunit binds to the mRNA and the anticodon end of the tRNAs and thus is most closely associated with the decoding function of the ribosome. The large subunit binds the acceptor ends of the tRNAs and is the site of peptide bond formation. These two fundamental activities of the ribosome—decoding and peptidyl transfer—must be integrated as the tRNA substrates ratchet their way through the interface region of the large and small subunits of the ribosome.

Our earlier work using primarily pre-steady state biochemical approaches focused on deciphering the molecular details of ribosome function during each step in the elongation cycle. What emerged was an increasing understanding of the ribosome as a dynamic machine that specifically responds to substrates as they bind—a "smart" ribosome. Such mechanisms are critical not only for tRNA selection but also for catalysis in the active site of the large ribosomal subunit and for release factor recognition of stop codons in the small ribosomal subunit. The ribosome appears to sample distinct conformational states that have essential roles in promoting catalysis (forward rate constants) and thus the translation cycle as a whole. In an exciting outcome from this work, we identified a novel mechanism for ensuring fidelity during protein synthesis following peptide bond formation. This quality control step relies on release factor–mediated abortive termination and increases the overall fidelity of translation at the level of both miscoding and frame-shifting. Ongoing work in the bacterial system uses biochemistry and ribosome profiling approaches to address fundamental features of termination and recycling, translational control, and quality control mechanisms. We are increasingly interested in how these pathways intersect with mRNA degradation, and thus broadly influence gene expression in these organisms.

Currently, the major focus of the laboratory is on translational control, mRNA surveillance, and events relevant to translational elongation and termination in eukaryotes. Again, we use pre-steady state kinetics and ribosome profiling to decipher mechanism in these systems. We have used biochemistry to implicate the termination factor homologs Dom34(PELO) and Hbs1(HBS1L) in destabilizing the subunit interface and in concert with the ATPase Rli1(ABCE1) in recycling ribosomes following termination or ribosome rescue. These biochemical results were supported by ribosome profiling studies in Dom34D and Rli1-degron strains revealing ribosome occupancy in unanticipated regions (the 3’ UTR and at sites of truncated mRNAs). Ribosome profiling studies in platelets and reticulocytes have revealed striking 3’ UTR occupancy which we have attributed to down regulation of PELO and ABCE1 in these tissue types. We have also implicated the DEAD-box helicase Dhh1(DDX6) in monitoring ribosome speed and, at least in part, specifying the rates of mRNA degradation in yeast. We are interested in determining at a molecular level how speed is discerned and how this information is communicated to the decay machinery. Ongoing biochemical and profiling studies will be used to define the molecular mechanics of ribosome function and its regulation. More broadly, these studies will help us to understand these key steps in gene expression that are critical to human health and disease.

Grants from the National Institutes of Health supported aspects of this work.

As of April 25, 2016

Scientist Profile

Investigator
The Johns Hopkins University
Biochemistry, Molecular Biology