Bacterial RNA polymerase and a “sigma” transcription factor form an initiation-competent “open” complex at a promoter by disruption of about 14 base pairs. Strand separation is likely initiated at the highly conserved -11 A-T base pair. Amino acids in conserved region 2.3 of the main E. coli sigma factor, σ70, are involved in this process, but their roles are unclear. To monitor the fates of particular bases upon addition of RNA polymerase, promoters bearing single substitutions of the fluorescent A-analog 2-AP at −11 and two other positions in promoter DNA were examined. Evidence was obtained for an open intermediate on the pathway to open complex formation, in which these 2-AP are no longer stacked onto their neighboring bases. The tyrosine at residue 430 in region 2.3 of σ70 was shown to be involved in quenching the fluorescence of a 2-AP substituted at −11, presumably through a stacking interaction. These data refine the structural model for open complex formation and reveal a novel interaction involved in DNA melting by RNA polymerase.

Formation of the stable, strand separated, ‘open’ complex between RNA polymerase and a promoter involves DNA melting of approximately 14 base pairs. The likely nucleation site is the highly conserved −11A base in the non-template strand of the −10 promoter region. Amino acid residues Y430 and W433 on the σ70 subunit of the RNA polymerase participate in the strand separation. The roles of −11A and of the Y430 and W433 were addressed by employing synthetic consensus promoters containing base analog and other substitutions at −11 in the non-template strand, and σ70 variants bearing amino acid substitutions at positions 430 and 433. Substitutions for −11A and for Y430 and W433 in σ70 have small or no effects on formation of the initial RNA polymerase-promoter complex, but exert their effects on subsequent steps on the way to formation of the open complex. As substitutions for Y430 and W433 also affect open complex formation on promoter DNA lacking the −11A base, it is concluded that these amino acid residues have other (or additional) roles, not involving the −11A. The effects of the substitutions at −11A of the promoter and Y430 and W433 of σ70 are cumulative.

Formation of strand-separated, functional complexes at promoters was compared for RNA polymerases from the mesophile Escherichia coli and the thermophile Thermus aquaticus. The RNA polymerases contained sigma factors that were wild type or bearing homologous alanine substitutions for two aromatic amino acids involved in DNA melting. Substitutions in the A subunit of T. aquaticus RNA polymerase impair promoter DNA melting equally at temperatures from 25 to 75°C. However, homologous substitutions in 70 render E. coli RNA polymerase progressively more melting-defective as the temperature is reduced below 37°C. The effects of the mutations on the mechanism of promoter DNA melting were investigated by studying the interaction of wild type and mutant RNA polymerases with "partial promoters" mimicking promoter DNA where the nucleation of DNA melting had taken place. Because T. aquaticus and E. coli RNA polymerases bound these templates similarly, it was concluded that the different effects of the mutations on the two polymerases are exerted at a step preceding nucleation of DNA melting. A model is presented for how this mechanistic difference between the two RNA polymerase could explain our observations. Transcription in bacteria is catalyzed by DNA-dependent RNA polymerase (RNAP), 1 a key enzyme for gene expression and its regulation (1, 2). Specific recognition of promoter DNA is mediated by an initiation subunit (sigma factor). The functional RNAP "holoenzyme" results when the sigma factor binds to the catalytic component, the "core" enzyme. A highly conserved region of 18 amino acids (designated region 2.3) of the main bacterial sigma factors has been shown to play a role in the melting process (3-6). Aromatic amino acid side chains in this region are positioned on the same side of an ␣ helix, poised to interact with the exposed bases of the promoter DNA (7,8).Studies with Escherichia coli (Eco) RNAP have indicated that upon RNAP binding to promoter DNA, an unstable closed complex (RP c ) is formed, followed by at least two additional intermediates (I 1 and I 2 ) before the initiation-competent, stable open complex (RP o ) is formed (9, 10), in which a 14-bp region of the promoter DNA, including the start site of transcription (2), has been melted. R ϩ P 7 RP c 7 I 1 7 I 2 7 RP o SCHEME 1In addition to the promoter DNA, the RNAP is thought to undergo conformational changes as well (9, 10).The past 6 years have seen a dramatic increase in structural information for bacterial RNAP, primarily because of the successful crystallization of the RNAP from two thermophilic bacteria, Thermus aquaticus (Taq) and Thermus thermophilus (8,(11)(12)(13)(14). The RNAP from these two organisms share extensive homology with all subunits of Eco RNAP, including the primary sigma factors. Four amino acids are different between the DNA melting regions (2.3) of Taq A and Eco 70 , but in three of the four cases the differences involve amino acids with chemically similar side chains. It is not well understood to what extent the mechanism of...

Initiation of transcription is an important target for regulation of gene expression. In bacteria, the formation of a transcription-competent complex between RNA polymerase and a promoter involves DNA strand separation over a stretch of about 14 base pairs. Aromatic and basic residues in conserved region 2.3 of Escherichia coli sigma(70) had been found to participate in this process, but it is still unclear which amino acid residues initiate it. Here we report an essential role for threonine (T) at position 429 of sigma(70): its substitution by alanine (T429A) results in the largest decrease in open complex formation yet observed for any single substitution in region 2.3. Promoter recognition itself is not affected by T429A substitution, thus providing evidence for a role of T429 in the strand-separation step. Our data are consistent with a model where the T429 would act as a competitor for the hydrogen bonding that stabilizes the highly conserved -11A-T base pairs of the promoter DNA, thus facilitating initiation of strand separation at this particular position in the -10 region. This model suggests an active role for RNA polymerase in disrupting the -11 base pair, rather than just capturing the -11A subsequent to spontaneous unpairing.