To achieve its developmental cycle, bacteriophage T4 takes over the RNA polymerase of its host, E. coli. E. coli RNA polymerase, like all bacterial polymerases, is composed of a core of subunits (beta, beta', alpha1, alpha2, and omega), which have RNA synthesizing activity, and a specificity factor (sigma), which 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. 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, AsiA-induced remodeling is proposed to make a surface accessible for MotA to bind to sigma70 region 4 in a process called sigma appropriation. X-ray crystallographic structures of bacterial polymerases predicts that the C-terminus of sigma70 (residues 600 to 613, H5) is intimately associated with the beta-flap. We have previously shown that H5 is also the binding site for MotA. We have continued our investigation how H5 contacts MotA and the beta-flap by using an E. coli 2-hybrid analysis to compare how various single substitutions within H5 affect its interactions with these two partmers. We have found that a shared set of residues (L607, R608, F610, L611 and D613) is required for either interaction. Previous workers isolated an E. coli strain, tabG, that does not support a T4 motA- infection, but otherwise grows normally;they mapped the tabG mutation in or near rpoB, which encodes the beta subunit of polymerase. We have determined that tabG has two rpoB mutations, E835K and G1249D. Working with T. Cardozo (New York University), we modeled these residues on available polymerase structures. E835 is at the base of the -flap;G1249 is immediately adjacent to a feature called switch 3 loop, where the extruding RNA enters the RNA exit channel. We transduced the rpoB mutations into BL21(DE3), generating the strain B11. Like tabG, B11 restricts T4 motA- growth, and complementation assays indicate that G1249D is responsible for the phenotype. Our primer extension analyses of RNA from wild type and motA- infections of B11 and BL21(DE3) have demonstated that MotA-dependent RNAs are modestly to significantly reduced in the B11 background, depending on the particular Pm. In motA- infections of the wild type host, there is a trace of Pm activity, presumably arising from the leakiness of the motA mutation. This is eliminated in B11, consistent with the inability of T4 motA- to grow in B11. Our results implicate a role for the switch 3 loop in activation by MotA. We speculate that this region of beta could affect sigma appropriation or promoter clearance at T4 middle promoters.