Bacteriophage T4 Gene Expression

Diabetes, Digestive, Kidney Diseases

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Abstract

Normal cell development requires regulation of transcription initiation and activation in order to express appropriate genes at appropriate times. We study regulation of transcription initiation using a simple prokaryotic system: E. coli RNA polymerase, a five subunit complex comprised of a core (2 alphas, beta, and beta prime) and a sigma specificity factor. The sigma70 factor specifies transcription from promoters that are responsible for basal gene expression during vegetative growth. When sigma70 is present within polymerase, two of its domains, 2.4 (an internal region) and 4.2 (the C-terminal region), interact with sequences within the minus 10 and minus 35 regions, respectively, of host promoter DNA. During infection by bacteriophage T4, this sequence specificity switches from host promoters to T4 middle promoters. Middle promoters contain the sigma70 recognition sequences at minus 10 but lack the canonical minus 35 sequences. Instead they have a 9 bp motif (a MotA box) which is centered at minus 30. Two phage proteins are required for this switch: the transcriptional activator MotA, which binds the MotA box and interacts with sigma70 and the T4 co-activator AsiA, which binds tightly to sigma70. Although AsiA is required as a co-activator for MotA-dependent transcription from T4 middle promoters, in the absence of MotA it is a potent inhibitor of transcription from sigma70 promoters. Our previous work has indicated that amino acids within the N-terminal half of AsiA, specifically V14, L18, and I40, are involved in forming or maintaining the AsiA/sigma70 complex and that the C-terminal region of AsiA aids inhibition by slowing the formation of transcription complexes between a promoter and AsiA/polymerase. To extend these studies, we have collaborated with the laboratory of Dr. Eric Miller (North Carolina State University) to investigate a predicted asiA gene in the genome of KVP40, a T4-related phage that infects V. cholerae and other Vibrio species. The sequence of the 99 amino acid KVP40 gene product (derived from base coordinates 9,539 to 9,835 of the 245 kbp genome) is 32% identical to that of the 90 amino acid T4 AsiA protein, between T4 residues 10 and 76. We have cloned the KVP40 gene into vectors suitable for protein expression and 2-hybrid analyses. In an E. coli 2-hybrid system, we find that the KVP40 protein, like T4 AsiA, interacts with region 4 of E. coli sigma70, a region that is nearly identical in amino acid sequence to region 4 of the primary sigma factor of V. cholerae. Production of the KVP40 protein, like the production of T4 AsiA, is toxic to E. coli, arresting growth within about an hour of protein induction. In in vitro transcription analyses using the T4 middle promoter PuvsX, the KVP40 protein is able to inhibit transcription nearly completely in the absence of MotA and to activate transcription in the presence of MotA. Our results are consistent with the assignment of the KVP40 protein as a T4 AsiA ortholog and suggest that like T4, KVP40 uses AsiA as a transcriptional inhibitor and co-activator. Curiously, no MotA protein ortholog or MotA-dependent promoters are identified in the KVP40 genome. Our results suggest that KVP40 encodes an unusual MotA protein or that the co-activation function of the KVP40 AsiA provides a novel function for the phage that is yet to be determined. E. coli itself also encodes an anti-sigma70 factor, the Rsd protein. Previous work in other labs has indicated that Rsd interacts with region 4.2 of sigma70 and inhibits transcription from some E. coli sigma70 promoters. To investigate further its functional similarity with the AsiA proteins, we cloned the rsd gene and purified the expressed Rsd protein. Like T4 AsiA, Rsd forms a complex with sigma70 that is stable to electrophoresis and inhibits transcription from PuvsX. However, this inhibition is relatively weak, reducing the level of PuvsX RNA only about 2-fold. In addition, production of the Rsd protein slows, but does not arrest, E. coli growth, suggesting that it has less inhibition activity in vivo than does AsiA. Rsd also does not act as a co-activator of MotA-dependent transcription. Our results suggest that while Rsd, the T4 AsiA protein, and the KVP40 AsiA share common functional features, only the phage AsiA proteins can provide a co-activation function with MotA.