Molecular Mechanisms of Translation and Their Implications for Gene Regulation
Summary: Rachel Green uses primarily biochemical 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, known as 30S in bacteria, 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, known as 50S in bacteria, 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.
We use primarily biochemical approaches to study the molecular mechanism of translation. We have been interested in defining the role played by the rRNAs and tRNA in these processes. How do the nucleotides in the active site conspire in the highly regulated formation of peptide bonds? How is peptide bond formation linked to intersubunit interaction and movement? What are the relative movements of the active-site components during the translational cycle? How is high-fidelity tRNA selection facilitated through the actions of the GTPase elongation factor EF-Tu? How is movement of the tRNA:mRNA complex promoted by the GTPase EF-G? How is high-fidelity peptide release stimulated by the protein release factors?
An emerging theme in the translation field is that the ribosome is a dynamic machine that specifically responds to substrates as they bind—a "smart" ribosome. Such mechanisms appear to be 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. We focus on deciphering the mechanisms at the heart of each step in the translation cycle, using pre-steady-state biochemical and biophysical approaches to identify the molecular components that are involved and how they each contribute. Our recent work in this general area has 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. We are currently focused on understanding the in vivo relevance of this system and its implications for the quality and yield of overexpressed proteins in bacterial and eukaryotic systems.
In other new studies, we are moving into the area of translation control mechanisms in bacteria and in eukaryotes, particularly in systems where gene regulation might take place at a post-initiation stage. We anticipate that our earlier work on the core mechanisms of ribosome function will guide our work on gene regulation wherein built-in modes of control in the ribosome are targeted by the vast array of extraribosomal factors. Current projects in these areas focus on the regulation of translation by peptide-mediated stalling, by microRNAs, and by protein factors essential for nonsense-mediated and no-go decay. We have recently reconstituted eukaryotic translation in vitro (using purified yeast components), thus paving the way for more detailed investigations in a broad range of areas related to translational control.
Grants from the National Institutes of Health supported aspects of this work.
As of May 30, 2012