Role of Interactions of the CRE Region of Escherichia coli RNA Polymerase with Nontemplate DNA during Promoter Escape

RNA polymerase (RNAP) recognizes promoter DNA through many interactions that determine specificity of transcription initiation. In addition to the dedicated transcription initiation σ factor in bacteria, the core enzyme of RNAP can also participate in promoter recognition. In particular, guanine residue at the +2 position (+2G) of the nontemplate DNA strand is bound in the CRE pocket formed by the RNAP β subunit. Here, we analyzed the role of these contacts in the process of promoter escape by RNAP by studying point mutations in the β subunit of Escherichia coli RNAP that disrupted these interactions. We found that the presence of +2G in the promoter slowed down the rate of promoter escape and increased proportion of inactive complexes. Amino acid substitutions in the CRE pocket decreased the promoter complex stability and changed the pattern of short RNA products synthesized during initiation, but did not significantly affect the rate of transition to elongation, regardless of the presence of +2G. Thus, the contacts of the CRE pocket with +2G do not make a significant contribution to the kinetics of promoter escape by RNAP, while the observed changes in the efficiency of abortive synthesis are not directly related to the rate of promoter escape.