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 has 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 those 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 (MotA NTD) to bind to the C-terminus of sigma70 and the C-terminal domain of MotA (MotA CTD) 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 an activator/RNA polymerase/DNA complex. However, the type of activation performed by this crystallized complex is fundamentally different from that of sigma appropriation. We combined biochemical analyses, available structures, and modeling to develop a structural model of sigma appropriation. 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. Our work suggested that MotA interacts with its DNA binding motif using a previously unidentified interaction mechanism in which the double wing helix structure of the CTD contacts the major groove of the DNA and the linker contacts the minor groove. In collaboration with the laboratory of Dr. Steve White (St Judes), we have now solved the crystal structure of the MotA linker-CTD with the DNA. Our results confirm that MotA CTD binds the DNA across the major groove via multiple interactions with the flanking sugar-phosphate backbones. It uses just two amino acids to read the MotA box motif, and the flexible N-terminal linker region interacts both specifically and non-specifically with the minor groove. In wild type T4 DNA, position 5 of cytosines are modified via hydroxymethylation/glucosylation. Out structure indicates that MotA CTD provides a cavity within the complex that is ideally positioned to accommodate this moiety and thereby increase the strength and specificity of the interaction. Using EMSAs and surface plasmon resonance, we have demonstrated that DNA containing the beta modification significantly enhances MotA binding affinity, primarily by decreasing the off-rate. Our results indicate that the double-wing domain of MotA represents an entirely new way in which a small protein module can recognize and bind DNA.
