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. To understand sigma appropriation at a molecular level, it is important to know how MotA interacts with the DNA. However, while there are separate structures for MotANTD and MotACTD, attempts to obtain a structure of full length MotA with or without the DNA have been unsuccessful. Consequently, to generate a map of the MotA/DNA interaction within the sigma-appropriated complex, we performed DNA cleavage reactions with iron bromoacetamidobenzyl-EDTA (FeBABE), which we covalently attached to cysteines introduced into MotA. 32P-labeled DNA was cleaved by the induction of the Fenton reaction that generates OH- radicals near the chelate. The generated cut sites were evaluated using ICM Molsoft and 3D physical models of MotANTD, MotACTD, and DNA to guide the position of MotA;we modeled the linker using Molsoft modeling. We found that the unusual double wing motif present in MotACTD resides in the major groove of the MotA box while the C-terminal portion of MotANTD is near the upstream minor groove. Thus, we have positioned full-length MotA within the transcription complex and demonstrated how this approach can map a protein/DNA complex that has been recalcitrant to traditional structure analyses. In addition, our Surface Plasmon Resonance studies found that MotA alone is in a dynamic equilibrium with the MotA element. Our results are consistent with a model in which MotA samples the DNA and is able to activate only when its binding sites on sigma70 and the DNA are both available.

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James, Tamara D; Cardozo, Timothy; Abell, Lauren E et al. (2016) Visualizing the phage T4 activated transcription complex of DNA and E. coli RNA polymerase. Nucleic Acids Res 44:7974-88
Hsieh, Meng-Lun; James, Tamara D; Knipling, Leslie et al. (2013) Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation. J Biol Chem 288:27607-18
Decker, Kimberly B; Hinton, Deborah M (2013) Transcription regulation at the core: similarities among bacterial, archaeal, and eukaryotic RNA polymerases. Annu Rev Microbiol 67:113-39
James, Tamara D; Cashel, Michael; Hinton, Deborah M (2010) A mutation within the {beta} subunit of Escherichia coli RNA polymerase impairs transcription from bacteriophage T4 middle promoters. J Bacteriol :