Normal cell development requires regulation of transcription initiation and activation in order to express appropriate genes at appropriate times. We study regulation of transcription initiation using a simple prokaryotic system: E. coli RNA polymerase, a five subunit complex comprised of a core (2 alphas, beta, and beta') and a sigma specificity factor. The sigma70 factor specifies transcription from promoters that are responsible for basal gene expression during vegetative growth. When sigma70 is present within polymerase, two of its domains, 2.4 (an internal region) and 4.2 (the C-terminal region), interact with sequences within the -10 and -35 regions, respectively, of host promoter DNA. During infection by bacteriophage T4, this sequence specificity switches from host promoters to T4 middle promoters. Middle promoters contain the sigma70 recognition sequences at -10 but lack the canonical -35 sequences. Instead they have a 9 bp motif (a MotA box) which is centered at -30. Two phage proteins are required for this switch: the transcriptional activator MotA, which binds the MotA box and interacts with sigma70 and the T4 co-activator AsiA, which binds tightly to sigma70. Although AsiA is required as a co-activator for MotA-dependent transcription from T4 middle promoters, it also functions as an transcriptional inhibitor in the absence of MotA. Our previous work has indicated that both MotA and AsiA interact within region 4 of sigma70. By using a 2-hybrid assay, we have identified amino acids within the far C-terminal region of sigma70 that are needed for the MotA/sigma70 interaction. Our in vitro transcription experiments indicate that a polymerase reconstituted with a sigma70 that lacks the C-terminal amino acids 604-613 or 608-613 is defective for MotA-dependent activation. These results support our previously suggested model that that the protein-protein interactions of MotA and AsiA with region 4 of sigma70 serve to substitute functionally for an interaction between sigma70 and the -35 element of promoter DNA. To investigate which regions within the 90 amino acid AsiA protein are involved in forming a complex with sigma70 and in providing transcriptional functions we generated random mutations throughout AsiA and targeted mutations within the C-terminal region of the protein. We tested mutant proteins for their ability to complement the growth of T4 asiA am phage under nonsuppressing conditions, to inhibit the growth of E. coli, to interact with sigma70 region 4 in a 2-hybrid assay, to bind to sigma70 in a native protein gel, and to inhibit or activate transcription in vitro. Our results show that substitutions within the N-terminal half of AsiA, at amino acids V14, L18, and I40, rendered the protein significantly defective for binding to sigma70. These residues reside at the monomer/monomer interface that was recently reported in NMR structures of the AsiA dimer. In contrast, AsiA missing its C-terminal 44 amino acids interacted well with region 4 of sigma70 in the 2-hybrid assay, and AsiA missing its C-terminal 17 amino acids bound to sigma70 and was fully competent in standard in vitro transcription assays. However, the presence of the C-terminal region delayed the formation of transcriptionally competent species when the AsiA/polymerase complex was incubated with a promoter in the absence of MotA. Our results suggest that amino acids within the N-terminal half of AsiA are involved in forming or maintaining the AsiA/sigma70 complex. The C-terminal region of AsiA, while not absolutely required for inhibition or co-activation, aids inhibition by slowing the formation of transcription complexes between a promoter and the AsiA/polymerase complex.
