Rachel D. Green

Scholar: 1999

Awarded Institution
The Johns Hopkins University
Department of Molecular Biology & Genetics


Research Interests

Protein Synthesis: Ribosome-catalyzed peptidyl transferase; role of 23S rRNA in hybrid-states model for translation; in vitro selection of competent 23S rRNA derivatives

The ribosome, a two-subunit macromolecular complex, composed in bacteria of three large RNAs (rRNAs) and more than 50 proteins (r-proteins), is the catalyst and framework for the intricate and coordinated process of translation. We are interested in understanding the mechanism of ribosome-catalyzed peptidyl transferase and how this activity is controlled in the translational cycle. From an evolutionary perspective, we are focused on understanding whether 23S rRNA itself (rather than the ribosomal proteins) is directly involved in the catalysis of peptide bond formation.

In order to begin to define the architecture of the peptidyl transferase center of the ribosome, we have focused on identifying 23S rRNA elements proximal to the CCA acceptor ends of the A- and P-site bound tRNAs. In one study, we identified a specific nucleotide, G2252, that interacts in a Watson-Crick fashion with C74 of P-site bound tRNA. More recently, using a novel A site analogue crosslinking reagent, 4-thio-dT-p-C-p-Puromycin (s4TCPm) we identified another specific nucleotide, G2553, that is located proximal to the CCA end of A-site bound tRNA. Future experiments will focus on understanding how these elements function and move as the tRNAs ratchet their way through the ribosome during the translational cycle.

The hybrid states model for translation proposes that the acceptor and anticodon ends of the tRNA substrates move independently with respect to the ribosome during the translational cycle. First, the acceptor ends of the A and P site tRNAs translocate with respect to the 50S subunit as a result of peptidyl transferase (PT). Second, EF-G catalyzes the translocation of the anticodon end of the tRNA and the associated mRNA with respect to the 30S subunit, thus activating the peptidyl-tRNA for PT. We are interested in defining the structural and functional differences between the so called "classical" and "hybrid" ribosomal complexes. A variety of approaches are being used to approach this problem including kinetic analysis of mutant tRNA substrates, chemical structural probing of the rRNAs and functional dissection of the crosslinked A site substrate (s4TCPm) ribosome complex described above.

Finally, we have recently transformed the ribosome into a cis catalyst - such that as a result of peptide bond formation the ribosome itself becomes modified - allowing for the application of in vitro selection technologies to the study of the ribosome. Using this system, we hope to identify a minimal RNA element, related to 23S rRNA, able to perform the peptidyl transferase reaction in the absence of the ribosomal proteins. Such a molecule would provide strong evidence for the central and fundamental role played by the rRNAs in the evolution of the ribosome (in a so called "RNA world") and would be amenable to structural approaches limited by the daunting size of the ribosome. Ultimately, this system will be used to evolve the substrate specificity of the ribosome to generate encoded non-peptidyl products useful for combinatorial screening approaches.