Multilevel regulation of gene expression in lambda and related bacteriophages.

Abstract

Bacteriophage $\lambda$ is a member of a family of temperate bacteriophages. A number of interactions between $\lambda$ and E. coli host factors are important for phage gene expression and gene product action. Experiments examining the interactions between host proteins and phage proteins are presented in this thesis. The first section of the thesis describes structure/function studies of two related transcription elongation factors, the N and Nun proteins of phages $\lambda$ and HK022, respectively. The $\lambda$ N protein interacts with RNA polymerase and several E. coli host factors, called Nus factors, at the $\lambda\ nut$ site to allow antitermination of transcription. An N-modified transcription complex is able to override $\lambda$ transcription termination signals that would otherwise impede transcript elongation. The HK022 Nun factor, in conjunction with the same host factors and the $\lambda\ nut$ site, has the opposite effect; Nun terminates $\lambda$ transcription near the nut site. Chimeric proteins that combine regions of N and Nun were employed to determine which region(s) confer antitermination and termination activity. A protein containing amino acids 31-107 from N has antitermination activity, indicating that this region of N is necessary for antitermination activity. A protein containing amino acids 55-112 from Nun has specific termination activity, indicating that this region of Nun is necessary for termination activity. The second section of the thesis describes the identification of a phage $\phi$80 gene, rha, which inhibits phage growth in E. coli integration host factor (IHF) mutants. IHF is a histone-like DNA binding protein which has pleiotropic effects on gene expression. To elucidate the roles of Rha and IHF in the inhibition of phage growth, the $\phi$80 rha gene was sequenced, the developmental pattern of Rha protein production was determined, and a functional homologue of rha in the lambdoid phage P22 was identified. During infection of an IHF$\sp+$ host, Rha is produced only late in infection. By contrast, infection of an IHF-host results in some Rha production at early timepoints and much higher levels at later timepoints. IHF influences both the timing and amount of Rha production.