One of the potential regulatory steps in procaryotic transcription is promoter clearance, a transition step in transcription initiation at which an RNA polymerase (RNAP) switches from the initial, transcribing stage to the elongation stage. The biological significance of promoter clearance and the role of RNAP in this process are not understood. One approach to address these questions is to study mutant RNAPs that have altered promoter clearance. Because the antibiotic rifampicin inhibits transcription by preventing an initial transcribing complex from entering the elongation mode, mutant RNAPs which confer rifampicin (Rif(r)) are likely to be altered in promoter clearance. To test this hypothesis, we studied the effects of Rif(r) RNAPs on the pyrBI promoter, which is subject to control of promoter clearance in response to the availability of UTP. Two Rif(r) alleles that carry a different altered amino acid residue at position 529 of the β subunit appeared to be defective in transcription from the pyrBI promoter in vivo. Biochemical analysis of one of these mutant RNAPs, RpoB3401 with a R529C change in the β subunit, showed that it overproduces aborted initiation products from the pyrBI promoter and thus is defective in promoter clearance leading to reduced productive initiation. The severity of overproducing the aborted initiation products is an inverse function of the UTP concentration indicating that RpoB3401 has reduced affinity for UTP and thus is subject to a high Km barrier during promoter clearance. The defect of RpoB3401 in abortive initiation in vitro could account fully for its reduced initiation activity in vivo demonstrating the biological significance of abortive synthesis in transcription initiation. Our results indicate that at least part of the ’rif region’ is important for the process of abortive initiation and that promoter clearance can be regulated in part by modulating the Km of RNAP for nucleotides during initiation. The mutant enzyme is not altered in stuttering RNA synthesis at the pyrBI promoter, previously observed with wild-type RNAP. Our results also show that the mechanisms underlying the two non-productive initiation events (abortive synthesis and stuttering synthesis) at the pyrBI promoter are distinct. © 1994 Academic Press Limited.