The mechanism of RNA cleavage by the hammerhead ribozyme and the sequence specific """"""""recognition"""""""" of RNA by bacteriophage coat proteins will be studied by biochemical and biophysical methods. The two projects were chosen because they allow a detailed study of RNA function in a situation where the biologically relevant activity is contained with an RNA sufficiently small that variants can easily be synthesized by chemical or embryological methods. The availability of several X-ray crystal structures and quantitative assays for both systems permits the design of sophisticated experiments to refine our concepts of how RNA """"""""works"""""""". Experiments on the hammerhead will focus on obtaining additional evidence that the X-ray structure and the major solution conformation are not in a catalytically active conformation. A nucleotide analogue interference approach will be used to identify essential functional groups and attempt to identify revertants of hammerhead base mutations. Hammerhead modifications that introduce steric bulk and phosphorodithioates will also be studied. Site directed crosslinking and in vitro selection strategies are proposed to stabilize in quite different active conformation that normally is believed to form only transiently. Hammerhead cleavage in crystals will be compared to the reaction in solution. Experiments on MS2 and Qbeta phage coat protein will focus on understanding how they interact with RNAs that differ from their major target. The possibility that MS2 coat protein can bind RNA in two different binding modes will be investigated. The distantly related Qbeta system will be investigated with the glial of understanding how certain mutants in either protein cause relaxed specificity. Finally, in vitro selection will be used to find RNAs of very different structure that bind MS2 coat protein in a way that differs from the major target.
Showing the most recent 10 out of 51 publications
