This collaborative project is directed at designing, synthesizing and characterizing a new generation of crosslinkable derivatives of ribo and deoxyribonucleotides to be used for structural probing of a variety of polymerizing enzymes, including DNA and RNA polymerases of prokaryotic, eukaryotic and viral origin. The approach is based on the highly selective affinity labeling technique developed by the Russian colleagues participating in the collaboration. Two classes of compounds will be synthesized: (a) with reactive group positioned on the gamma-phosphate and (b) with substitutions in the base ring of uracil. In each class, crosslinking reagents specific to different amino acids, with different mechanisms of activation, and with varying length of the reagent """"""""arm"""""""" will be prepared. Such types of nucleotide derivatives can be utilized as substrates during in vitro enzymatic syntheses of DNA and RNA. Thus, the reagents synthesized can be used for in vitro crosslinking to the substrate or product binding site of the polymerase under study followed by extension with the next radiolabeled nucleotide resulting in a radioactive nucleotide reporter group crosslinked to the protein. The site of the crosslinking can then be mapped using standard protein chemical approaches. This proposal represents an expansion of the currently active parent NIH grant (R0l GM30717) focused on site-directed mutagenesis of RNA polymerase genes in E. coli and functional analysis of the mutant enzymes. Mapping of functional sites using new affinity reagents will be invaluable for planning and interpreting the genetic experiments of the parent project.
