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 (rRNAs) and more than 50 proteins, is the catalyst and framework for the intricate and coordinated process of translation. Theoretical considerations, biochemical, genetic and now structural evidence indicate that the rRNAs play a central role in the processes of translation including aminoacyl tRNA loading, peptide bond formation, translocation of the mRNA:tRNA complex and peptide release. Our general goals are to define the molecular details of the translation process by focusing on the role played by the rRNAs in each of these basic steps. While structural information has provided us with candidates regions that may play direct roles in catalysis and movement, we are unable to define their specific role in the absence of biochemical dissection. Further, the dynamics of the translation cycle have been difficult to extract from the static views that we obtain from X-ray crystallography and cryoEM approaches. This proposal outlines a series of experiments focused on using pre-steady state kinetic analysis to define the contributions of specific rRNA elements to the different steps in translation. Site-directed mutagenesis will target critical regions and an in vivo tagging system will be used to allow for the isolation of variant ribosomes in vitro. Stopped-flow fluorescence will be used to monitor tRNA binding, release factor interactions and translocation while rapid quench approaches will be used to study the rates of GTP hydrolysis, peptide bond formation and peptide release. Chemical modification experiments and genetic screens are also proposed to assist in the identification of other regions of the rRNA that are critical in the dynamics of translation. Information obtained from such studies will help in defining the features of current antibiotics that make them effective and ultimately in the design of the new classes of therapeutic compounds. ? ?
| Schuller, Anthony P; Wu, Colin Chih-Chien; Dever, Thomas E et al. (2017) eIF5A Functions Globally in Translation Elongation and Termination. Mol Cell 66:194-205.e5 |
| Buskirk, Allen R; Green, Rachel (2017) Ribosome pausing, arrest and rescue in bacteria and eukaryotes. Philos Trans R Soc Lond B Biol Sci 372: |
| Petropoulos, Alexandros D; McDonald, Megan E; Green, Rachel et al. (2014) Distinct roles for release factor 1 and release factor 2 in translational quality control. J Biol Chem 289:17589-96 |
| Koutmou, Kristin S; McDonald, Megan E; Brunelle, Julie L et al. (2014) RF3:GTP promotes rapid dissociation of the class 1 termination factor. RNA 20:609-20 |
| Guydosh, Nicholas R; Green, Rachel (2014) Dom34 rescues ribosomes in 3' untranslated regions. Cell 156:950-62 |
| Green, Rachel; Rogers, Elizabeth J (2013) Transformation of chemically competent E. coli. Methods Enzymol 529:329-36 |
| Buskirk, Allen R; Green, Rachel (2013) Biochemistry. Getting past polyproline pauses. Science 339:38-9 |
| He, Shan L; Green, Rachel (2013) Northern blotting. Methods Enzymol 530:75-87 |
| Djuranovic, Sergej; Nahvi, Ali; Green, Rachel (2012) miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay. Science 336:237-40 |
| Shoemaker, Christopher J; Green, Rachel (2012) Translation drives mRNA quality control. Nat Struct Mol Biol 19:594-601 |
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