Different roles for basic and aromatic amino acids in conserved region 2 of Escherichia coli sigma(70) in the nucleation and maintenance of the single-stranded DNA bubble in open RNA polymerase-promoter complexes.

Amino acid residues in region 2 of final sigma(70) have been shown to play an important role in the strand separation step that is necessary for formation of the functional or open RNA polymerase-promoter complex. Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2. We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with single-stranded DNA, and with forked DNA duplexes carrying a single-stranded overhang consisting of bases in the -10 region. We concluded that two basic amino acids (Lys(414) and Lys(418)) are important for promoter binding and demonstrated distinct roles, at a subsequent step, for two aromatic amino acids (Tyr(430) and Trp(433)). It is likely that these four amino acids, which are close to each other in the structure of final sigma(70), together are involved in the nucleation of the strand separation process.

Aromatic amino acids in region 2.3 of Escherichia coli sigma 70 participate collectively in the formation of an RNA polymerase-promoter open complex.

Formation of an initiation-competent RNA polymerase-promoter complex involves DNA melting over a region of about 12 base-pairs, which includes the start site of transcription, thus enabling the template strand to base-pair with the initiating nucleoside triphosphates. By studying the effects of alanine substitutions, we have investigated the role of the aromatic amino residues in the Escherichia coli sigma(70) conserved region 2.3 in promoter strand separation. The resulting mutants were assessed for their activity in vivo in the context of a sigma(70)/sigma(32) hybrid sigma factor that could be targeted to a specific hybrid promoter in the cell. All substitutions lead to an at least twofold reduction in expression of the hybrid promoter-driven reporter gene. The in vitro assay of single substitutions indicated cold sensitivity similar to that previously observed with analogous substitutions in Bacillus subtilis sigma(A). Kinetic assays showed that these substitutions slowed the rate of open complex formation at 37 degrees C as well. RNA polymerase reconstituted with a sigma(70) containing multiple alanine substitutions readily binds to promoter DNA, but then proceeds slowly beyond the first intermediate complex on the pathway to formation of the transcription-competent complex. These data demonstrate that together the aromatic residues in region 2.3 of E. coli sigma(70) ensure that DNA strand separation proceeds efficiently, even if no individual residue may be essential for accomplishment of the process.