One class of genetic regulatory mechanism involves direct interaction of regulatory factors with an RNA polymerase leading to changes in the specificity of transcription. Our studies approach the identification and biochemical dissection of this king of process by (1) elucidating the mechanisms of the basic steps in transcription in vitro; (2) developing in vitro systems in which the efficiency and selectivity of transcription parallels the cellular process. The basic mechanisms involved in the elongation and termination of RNA chains are not yet well understood. We propose to study the reaction in which RNA polymerase pauses at certain sequences during elongation or termination. Using DNA from deletion mutants of T7 phage, or the plasmid pKK3535 bearing the rrnB operon, we will determine the nucleotide sequences of pause sites and the effect of regulatory factors that alter the pausing process such as ppGpp, nusA protein and tho factor. DNa template having single base changes in the E. coli trp attenuator locus will be used to determine whether pausing at a terminator/attenuator results from formation of an RNA hairpin structure or is determined by sequences in the non-transcribed DNA strand. Several lines of evidence suggest that transcriptional elongation in vivo involves factors in addition to RNA polymerase and takes place on nucleoprotein templates. We will study transcription which takes place on endogenous nucleoprotein templates in cell extracts to develop an in vitro transcription system in which in vivo rates of transcription are achieved. The effect of regulatory factors such as ppGpp and mutations that alter RNA synthesis in vivo will be tested. Vegetative cells of B. subtilis contain an RNA polymerase (Sigma28 RNA polymerase) which utilizes a set of strong promoter sites on the B. subtilis genome not used by the normal holoenzyme. We propose to study the synthesis and function of Sigma28 in B. subtilis during normal growth and sporulation. The sequence of representative strong promoter sites for B. subtilis RNa polymerase II will be determined, and we will use chemical probes to study the interaction of holoenzyme II with its cognate promotor sites.
