The National Institute of Diabetes & Digestive & Kidney Diseases

Mechanisms of Transcription Initiation and Activation: A Simple Model System Based on Bacteriophage T4 Gene Expression

Regulation of transcription, which is critical for normal development, is often achieved by the action of transcriptional activators and coactivators. We study a simple prokaryotic system to investigate activation of transcription initiation. Our work has defined the proteins and DNA sequences needed for the activation of a class of bacteriophage T4 promoters. We have shown that a phage activator and coactivator interact with the sigma-70 subunit of E. coli RNA polymerase, the subunit that defines the DNA sequence specificity of the polymerase. Through their interactions with sigma, these phage proteins inhibit transcription from typical E. coli promoter sequences while directing the polymerase to new phage promoters. This type of activation represents a novel class of activation in prokaryotes. In addition, it resembles the action of certain eukaryotic transcription factors that work by binding to the major specificity factor of eukaryotic polymerases. Thus, this simple phage system can provide insight into how this type of activation works in higher organisms. In addition, understanding how the phage proteins inhibit host transcription my reveal new antibacterial strategies.

Our current efforts involve an analysis of the phage system at a molecular level. Through our construction and biochemical characterization of mutant activator and coactivator proteins, we are investigating how particular protein domains and specific amino acids contribute to the activation of phage promoter transcription and the inhibition of host promoter transcription. This work has been aided by recent structural analyses of the phage proteins and E. coli sigma-70. Our goal is to determine how the protein-protein and protein-DNA interactions within the activator/coactivator/RNA polymerase/promoter complex direct the polymerase to recognize new promoter sequences.

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2. Baxter K, Lee J, Minakhin L, Severinov K, Hinton DM Mutational analysis of sigma70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA. J Mol Biol (363): 931-44, 2006. [Full Text/Abstract]

3. Hinton DM, Vuthoori S, Mulamba R The bacteriophage T4 inhibitor and coactivator AsiA inhibits Escherichia coli RNA Polymerase more rapidly in the absence of sigma70 region 1.1: evidence that region 1.1 stabilizes the interaction between sigma70 and core. J Bacteriol (188): 1279-85, 2006. [Full Text/Abstract]

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5. Hinton DM, Pande S, Wais N, Johnson XB, Vuthoori M, Makela A, Hook-Barnard I Transcriptional takeover by sigma appropriation: remodelling of the sigma70 subunit of Escherichia coli RNA polymerase by the bacteriophage T4 activator MotA and co-activator AsiA. Microbiology (151): 1729-40, 2005. [Full Text/Abstract]

6. Pineda, M., Gregory, B. D., Szczypinski, B. Baxter, K. R., Hochschild,A., Miller, E. S., and Hinton, D. M. A Family of Anti-s70 Proteins in T4-type Phages and Bacteria That Are Similar to AsiA, a TranscriptionInhibitor and Co-activator of Bacteriophage T4. J. Mol. Biol. , 2004. [In Press]

7. Pal D, Vuthoori M, Pande S, Wheeler D, Hinton DM Analysis of regions within the bacteriophage T4 AsiA protein involved in its binding to the sigma70 subunit of E. coli RNA polymerase and its role as a transcriptional inhibitor and co-activator. J Mol Biol (325): 827-41, 2003. [Full Text/Abstract]

8. Pande S, Makela A, Dove SL, Nickels BE, Hochschild A, Hinton DM The bacteriophage T4 transcription activator MotA interacts with the far-C-terminal region of the sigma70 subunit of Escherichia coli RNA polymerase. J Bacteriol (184): 3957-64, 2002. [Full Text/Abstract]

9. Vuthoori S, Bowers CW, McCracken A, Dombroski AJ, Hinton DM Domain 1.1 of the sigma(70) subunit of Escherichia coli RNA polymerase modulates the formation of stable polymerase/promoter complexes. J Mol Biol (309): 561-72, 2001. [Full Text/Abstract]

10. Hinton DM, Vuthoori S Efficient inhibition of Escherichia coli RNA polymerase by the bacteriophage T4 AsiA protein requires that AsiA binds first to free sigma70. J Mol Biol (304): 731-9, 2000. [Full Text/Abstract]

11. Sharma M, Marshall P, Hinton DM Binding of the bacteriophage T4 transcriptional activator, MotA, to T4 middle promoter DNA: evidence for both major and minor groove contacts. J Mol Biol (290): 905-15, 1999. [Full Text/Abstract]

12. Marshall P, Sharma M, Hinton DM The bacteriophage T4 transcriptional activator MotA accepts various base-pair changes within its binding sequence. J Mol Biol (285): 931-44, 1999. [Full Text/Abstract]

13. Gerber JS Hinton DM An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription. J Bacteriol (178): 6133-9, 1996. [Full Text/Abstract]

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16. March-Amegadzie R Hinton DM The bacteriophage T4 middle promoter PuvsX: analysis of regions important for binding of the T4 transcriptional activator MotA and for activation of transcription. Mol Microbiol (15): 649-60, 1995. [Full Text/Abstract]