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 workers have found that T4 containing an amber (am) mutation within the motA gene, motA(am), or within the asiA gene, asiA(am), grows poorly in wild type E. coli;however, T4 motA(am) does not grow in the E. coli mutant strain, TabG. We have found that the RNA polymerase in TabG contains two mutations within its beta subunit gene: rpoB E835K and rpoB G1249D. Working with T. Cardozo (New York University) and the MolSoft program, we modeled these residues on available polymerase structures. E835 is at the base of the beta-flap while G1249 is immediately adjacent to a feature called the Switch 3 loop, where the extruding RNA enters the RNA exit channel. We transduced the rpoB mutations into BL21(DE3), generating the strain B11, and compared the growth of T4 asiA and motA mutants and the level of transcription from multiple T4 early and middle promoters in the rpoB wt strain BL21(DE3) and the rpoB mutant strain B11. While there is no significant growth phenotype for B11 in the absence of T4 infection, B11 restricts the growth of either T4 motA(am) or T4 asiA (am). Furthermore, during a T4 wt infection of B11, transcription from T4 middle promoters is specifically impaired. Using plasmids that express high levels of the beta protein that contains either wt E835 (but still G1249D) or wt G1249 (but still E835K), we have demonstrated that it is the G1249D mutation that is responsible for restricting the growth of either T4 motA(am) or asiA(am) and for impairing transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from tested T4 early promoters is either unaffected or in some cases, even increases. Our results indicate that the G1249D mutation specifically affects MotA/AsiA activation and suggest that the presence of MotA and AsiA may impair the function of the Switch 3 loop or that this portion of βmay influence interactions among MotA, AsiA, and RNA polymerase.

Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
2010
Total Cost
$797,697
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