Evidence for a tyrosine-adenine stacking interaction and for a short-lived open intermediate subsequent to initial binding of Escherichia coli RNA polymerase to promoter DNA.

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.

Threonine 429 of Escherichia coli sigma 70 is a key participant in promoter DNA melting by RNA polymerase.

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.

The -11A of promoter DNA and two conserved amino acids in the melting region of sigma70 both directly affect the rate limiting step in formation of the stable RNA polymerase-promoter complex, but they do not necessarily interact.

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 sigma70 subunit of the RNA polymerase participate in the strand separation.

Mechanistic differences in promoter DNA melting by Thermus aquaticus and Escherichia coli RNA polymerases.

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 sigmaA subunit of T.