NSF Award
9218217
RNA polymerase is the principal enzyme of gene expression and the target for genetic regulation. Its basic structure-functional features are highly conserved among all living organisms. The broad goal of this project is the understanding of the molecular mechanism of RNA polymerase basic function. To this end a combined genetic and biochemical approach will be taken using as model the RNA polymerase from Escherichia coli. The collaboration takes advantages of the biochemical strength of the U.S. side and specifically, it is proposed to generate an exhaustive collection of in-frame linker insertions and deletions in the cloned gene rpoB which encodes the B subunit of core RNA polymerase. The mutant RNA polymerase holoenzyme (a2BBo) will then be prepared for biochemical characterization by in vitro reconstitution from the individual subunits. The subunits will be obtained by overexpressing them from plasmids carrying the three rpo genes under the control of inducible promoter. The malfunctioning of RNA caused by mutations will be characterized using an arsenal of in vitro assays which would allow to monitor and quantitate individual steps of the enzyme's functional cycle. %%% This approach is anticipated to define and map the structural elements of the enzyme which carry out the multitude of RNA polymerase biochemical functions.
