The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of σ70 Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ70 and Core

RNA synthesis in Escherichia coli is accomplished by an RNA polymerase holoenzyme consisting of a core of five subunits (two α's, β, β′, and ω) and a σ specificity factor (reviewed in references 15 and 30). Core polymerase contains RNA synthesizing activity, but the specific start site for transcription initiation is determined by the DNA binding specificity of the particular σ factor. In E. coli, σ70 is the primary σ factor that is responsible for initiation of transcription during exponential growth. Sequence and structural analyses of various σ proteins from many prokaryotes indicate that primary σ factors share four main regions of conservation (regions 1 to 4) (15, 23). Sequence recognition capability in σ70 is provided by alpha helices within regions 2 and 4, which interact directly with a promoter's −10 and −35 DNA elements, respectively. Although most σ family members share conservation in regions 2 through 4, the sequence of region 1.1, the N-terminal, highly negatively charged portion, is conserved only among primary σ proteins (15, 23, 30). Within σ70, region 1.1 is composed of the first 100 residues. There is no evidence to suggest that region 1.1 interacts directly with promoter DNA. Nonetheless, its presence has a major impact on σ70-DNA interactions. In free σ70, the presence of region 1.1 prevents both specific and nonspecific binding to DNA (9). Early work suggested region 1.1 prevents this binding by directly interacting with region 4. However, subsequent work using nuclear magnetic resonance (NMR) spectroscopy of segmental, isotopically labeled σA of Thermotoga maritima, a σ70 analog, has failed to detect an interaction between regions 1.1 and 4.2 (5). Besides its function in free σ70, the presence of region 1.1 also modulates the ability of RNA polymerase holoenzyme to form stable, open complexes with certain promoters. Given the particular promoter, the presence of region 1.1 can either inhibit or promote the formation of the stable pretranscription complex (39, 40). Thus, region 1.1 provides important functions both when σ70 is free and when it is present within RNA polymerase. However, how region 1.1 works is not yet clear. The bacteriophage T4 AsiA protein has two functions. It inhibits σ70-dependent transcription from promoters that require an interaction between σ70 region 4 and the −35 DNA element and it coactivates transcription from T4 middle promoters that use σ70 containing RNA polymerase and the T4 transcription activator MotA (reviewed in reference 18). The MotA protein binds to a DNA motif (a MotA box) centered at position −30 of middle promoter DNA, and, in addition, both AsiA and MotA interact with σ70 region 4. A recent NMR structure of the AsiA/σ70 region 4 complex shows that AsiA contacts multiple σ70 residues, including residues that make direct contact with base moieties in the −35 element when RNA polymerase binds to a typical σ70-dependent promoter (22). Because the presence of region 1.1 modulates the interaction of σ70 with host promoter DNA and AsiA interacts with σ70 residues that contact the −35 element, we wondered whether σ70 region 1.1 was important for MotA/AsiA activation and AsiA inhibition. We show here that region 1.1 is not required for MotA/AsiA activation at a T4 middle promoter. However, RNA polymerase lacking region 1.1 is more rapidly inhibited by AsiA and more rapidly activated by MotA/AsiA. Our previous work indicated that AsiA binds efficiently to free σ70 but poorly, if at all, to holoenzyme (19). Our evidence supports the idea that AsiA inhibits more rapidly when region 1.1 is absent, because the absence of region 1.1 destabilizes the interaction of σ70 with core, resulting in more available free σ70 at any given time.