During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. This machinery is driven by E. coli RNA polymerase, which, like all bacterial polymerases, is composed of a core of subunits (beta, beta', alpha1, alpha2, and omega) that have RNA synthesizing activity and a specificity factor (sigma). The sigma protein identifies the start of transcription by recognizing and binding to sequence elements within promoter DNA. During exponential growth, the primary sigma of E. coli is sigma70, which, like all primary sigmas, is composed of four regions. Sigma70 recognizes DNA elements around positions -10 and -35 of host promoter DNA, using residues in its central portion (regions 2 and 3) and C-terminal portion (region 4), respectively. In addition, residues within region 4 must also interact with a structure within core polymerase, called the beta-flap, to position sigma70 region 4 so it can contact the -35 DNA. T4 takes over E. coli RNA polymerase through the action of phage-encoded factors that interact with polymerase and change its specificity for promoter DNA. Early T4 promoters, which have -10 and -35 elements that are similar to that of the host, are recognized by sigma70 regions 2 and 4, respectively. However, although T4 middle promoters have an excellent match to the sigma70 -10 element, they have a phage element (a MotA box) centered at -30 rather than the sigma70 -35 element. Two T4-encoded proteins, a DNA-binding activator (MotA) and a T4-encoded co-activator (AsiA), are required to activate the middle promoters. AsiA alone inhibits transcription from a large class of E. coli promoters by binding to and structurally remodeling sigma70 region 4, preventing its interaction with the -35 element and with the beta-flap. In addition to its inhibitory activity, the AsiA-induced remodeling allows the N-terminal domain of MotA (MotANTD) to bind to the C-terminus of sigma70 and the C-terminal domain of MotA (MotACTD) to bind to the MotA box. This process is called sigma appropriation. Despite dozens of activator crystallographic structures and recent RNA polymerase structures, there is only one complete structure of any activator/RNA polymerase/DNA complex. However, the type of activation performed by this crystallized complex is fundamentally different from that of sigma appropriation. We hypothesized that careful integration of the published structure of E. coli RNA polymerase with that of sigma70 Region 4 in complex with AsiA could result in a structural model of an AsiA-associated E. coli RNA polymerase. Furthermore, in previous work we had biochemically mapped the orientation of MotA relative to the DNA within the sigma-appropriated complex using MotA conjugated with the chemical cleaving reagent iron bromoacetamidobenzyl-EDTA (FeBABE). This work then provided a framework within which to incorporate our other analyses indicating how MotANTD interacts with sigma70. Rational combination of these heterogeneous data afforded a complete snapshot of the protein-protein and protein-DNA interactions within the sigma appropriation process. Our work has depicted how AsiA/MotA redirects sigma, and therefore RNA polymerase activity, to a T4 middle promoter DNA and how the flexibility of sigma Region 4 is likely crucial for this process. We had previously reported that a mutation within the beta subunit of RNA polymerase core, G1249D, specifically inhibits sigma appropriation although this mutation is far removed from the interaction sites of AsiA or MotA with polymerase. We have demonstrated that even though the mutated polymerase core is able to form a complex with AsiA/sigma, the presence of the mutation generates an AsiA-associated RNA polymerase that is much less competent for its interaction with MotA/DNA. Our model suggests that this arises because the glycine to aspartic substitution clashes with nearby negatively charged residues within sigma, impairing the flexibility needed for the AsiA-remodeled sigma Region 4 to contact MotA.
