The goals of this project are: (1) Determination of structural features of E. coli RNA polymerase (RNAP), particularly of its catalytic center and domains that interact with auxiliary factors, such as the various sigma factors. (2) Analysis of the biochemical mechanism of different steps of transcription by E. coli RNAP. (3) The screening and identification of antibiotics capable of inactivating rifampicin-resistant (Rifr) RNAP for potential clinical use. The realization of this goal will depend upon the structural and mechanistic studies of E. coli RNAP. Study of the nonproductive initiation has been focused. RNAP sometimes makes two kinds of nonproductive initiation products after the first phosphodiester bond formation and before entering into an elongation mode, which can be a rate-limiting step in transcription. One is abortive synthesis which is DNA-templated, the other is stuttering synthesis which is pseudo-templated. We have found that one Rifr RNAP mutant, RpoB3401, has reduced affinity for UTP and overproduced abortive initiation products at the pyrBI promoter. This result indicates the importance of Km of RNAP for nucleotide(s) in controlling the abortive initiation. We have found that wild-type RNAP also produces stuttering initiation products at the galP2 promoter. The stuttering synthesis at galP2 was sensitive to changes in UTP concentration and was repressed by transcription factor cAMP-CRP. A different Rifr RNAP mutant, RpoB3449, dramatically reduced its stuttering synthesis at galP2. These results indicate that the rif-region is important for both the abortive and stuttering synthesis in initiation. A Rifr mutation from pathogenic bacteria P. aeruginosa was sequenced and found to have the same amino acid change as that in E. coli indicating that the rif-region is highly conserved in bacteria. The target of several antibiotics including rifampicin is RNAP in bacteria. To test whether other antibiotics can inhibit Rifr RNAPs' function, we have determined the sensitivity of Rifr RNAPs to these antibiotics. Different degree of cross-resistance were found for different antibiotics. By this screening, we hope to identify and make suggestions in design of antibiotic for their ability to inhibit Rifr bacteria which has become a major problem clinically.
