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, AsiA-induced remodeling is proposed to make a surface accessible for MotA to bind to sigma70 region 4 in a process called sigma appropriation. Previous work has demonstrated that MotA contacts the carboxy-terminus of sigma70, a region that is normally bound within RNA polymerase by its interaction with the beta-flap tip. To identify the specific sigma70 residues responsible for interacting with MotA and the beta-flap tip, we generated single substitutions throughout the C-terminus of sigma70. We have found that MotA targets H5 residues that are normally engaged by the beta-flap. In 2-hybrid assays, the interaction of sigma70 with either the beta-flap tip or MotA is impaired by alanine substitutions at a set of identical residues (L607, R608, F610, L611, and D613). Transcription assays identify F610 and L611 as the key residues for MotA/AsiA-dependent transcription. F610 is a crucial residue in the H5/beta-flap tip interaction using promoter clearance assays with RNA polymerase alone. Our results show how the actions of small transcriptional factors on a defined local region of RNA polymerase can fundamentally change the specificity of polymerase.

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Patterson-West, Jennifer; James, Tamara D; Fernández-Coll, Llorenç et al. (2018) The E. coli Global Regulator DksA Reduces Transcription during T4 Infection. Viruses 10:
Cuypers, Maxime G; Robertson, Rosanna M; Knipling, Leslie et al. (2018) The phage T4 MotA transcription factor contains a novel DNA binding motif that specifically recognizes modified DNA. Nucleic Acids Res 46:5308-5318
Patterson-West, Jennifer; Arroyo-Mendoza, Melissa; Hsieh, Meng-Lun et al. (2018) The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness. Viruses 10:
Hinton, Deborah M (2017) A 3D puzzle approach to building protein-DNA structures. Transcription 8:113-119
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
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
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
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 :
Decker, Kimberly B; Hinton, Deborah M (2009) The secret to 6S: regulating RNA polymerase by ribo-sequestration. Mol Microbiol 73:137-40