The initiation of transcription is the point at which most bacterial genes are regulated. In bacteria, promoter specificity is partially determined by the sigma subunit of RNA polymerase. E. coli RNA polymerase (RNAP) is comprised of six subunits with DNA recognition provided by the sigma factor subunit σ⁷⁰ . σ⁷⁰ recognizes and activates canonical ‘housekeeping’ promoters. σ⁷⁰ possesses two DNA binding domains, regions 2 and region 4 which maintain specificity for the promoter elements at –10 and –35 relative to the transcriptional start site. These DNA recognition motifs are separable and independent and are connected by a flexible linker. Flexibility endows sigma factors with an ability to undergo a number of conformational changes important to their roles as activators.

Ancillary proteins bind sigma factors directly to attenuate transcription. The bacteriophage T4 provides an illustrative example. T4 AsiA is a versatile transcription factor that inhibits host gene expression as an ‘anti-sigma’ factor while promoting gene-specific expression of T4 middle genes in conjunction with T4 MotA. By binding conserved σ⁷⁰ region 4 (SR4), AsiA interferes with gene expression at promoters dependent on the –35 element. Remarkably, AsiA dismantles SR4, transforming the DNA-binding HTH element into a pseudo-continuous a-helix. Critical arginine residues for DNA binding in region 4.1 (R554 and R562) as well as region 4.2 (R584) are re-positioned away from the surface of EcSR4 where DNA would be located; these residues form new contacts with other SR4 regions to sequester them from the DNA. Other key residues for DNA interaction (e.g. T583) are buried at the AsiA/EcSR4 interface, leaving them inaccessible to the DNA in the complex. Thus, the structure of AsiA/EcSR4 demonstrates that AsiA blocks the DNA binding surface of SR4 almost in its entirety, complementing the blockage of core RNAP binding sites by AsiA on the domain.

The remodeling of SR4 by AsiA illustrates a different mechanism for an anti-sigma factor. Rather than reposition existing motifs using σ⁷⁰’s flexible linkers or occlude an existing surface, AsiA disrupts the three-dimensional fold of SR4.

The dramatic structural rearrangement in SR4 induced by AsiA explains why AsiA engages free σ⁷⁰ rather than σ⁷⁰ pre-associated with core polymerase. Since AsiA’s interaction surface on EcSR4 is coincident with the RNA polymerase binding site on region 4, it is logical that AsiA would prefer engagement with free σ⁷⁰. AsiA’s binding site on σ⁷⁰ is unobstructed in the free state, but would require competing with core polymerase binding surfaces in a pre-formed holoenzyme, something AsiA is inefficient at doing. This implies that engagement of a preformed AsiA/σ⁷⁰ complex with core polymerase would result in a remodeled holoenzyme, one where the position of AsiA/SR4 is altered on the surface of the enzyme.

We have established a novel mechanism by which a transactivating protein of bacteriophage can remodel a subunit of RNAP to inhibit expression of host genes and thereby impose promoter choice. The structure of AsiA/EcSR4 complex suggests that an AsiA-modified holoenzyme would have an altered position for region 4 on the enzyme surface without disruption of core polymerase interactions with other regions of σ⁷⁰. The conformation of altered polymerase conformation is currently under study.