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Bacteriophage T4 Gene Expression

Investigators
Hinton, Deborah
Institutions
DHHS/NIH - National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Start date
2007
End date
2007
Objective
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 (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. MotA is a two domain protein that that has been shown to interact with both region 4 of sigma70 and the MotA box

.In a collaboration with the laboratory of Dr. Milton Werner (Rockefeller University), we have defined the face of MotA that recognizes sigma70 region 4. NMR chemical shift analyses have indicated that MotA uses a basic/hydrophobic cleft to interact with the far C-terminus of the AsiA-remodeled sigma70, but MotA does not interact with AsiA itself. We have shown that mutations within this cleft (at MotA residues K3, K28, Q76, and S80) greatly reduce MotA interaction with sigma70 region 4, with the ability of MotA to activate transcription, and with phage viability. Previously described activators that target sigma70 directly have been found to employ acidic residues to engage a basic surface of region 4.

Our work supports accumulated evidence indicating that this sigma appropriation by MotA/AsiA is a fundamentally different mechanism of transcriptional activation.Because of our work with the T4 AsiA protein, we have also initiated an investigation of the Rsd (regulator of sigma D) protein of E. coli. The Rsd protein family is a highly conserved group of intracellular helical proteins with, at this point, largely undefined mechanisms of action. Rsd proteins are known to interact with sigma70 in a manner and location that could potentially inhibit the ability of RNA polymerase to activate transcription. This activity is often termed anti-sigma activity.

Rsd family proteins are found in many Gram-negative bacteria, including E. coli, Salmonella, Yersinia, Vibrio, and Pseudomonas species. Our previous biochemical and sequence analyses have suggested that there may be homology between the bacterial Rsd and phage AsiA families. To investigate this possibility, we have constructed a library of AsiA/Rsd chimera proteins, and assayed them for function. We find that a portion of Rsd that we previously proposed to be similar AsiA can substitute for that region of AsiA in an in vivo assay.

Funding Source
Nat'l. Inst. of Diabetes and Digestive and Kidney Diseases
Project number
1Z01DK057802-20
Categories
Escherichia coli
Salmonella