Translational Control of T4 Phage Gene Expression

点击翻译按钮获取中文摘要

The overall objective of this project is to elucidate the biological roles and mechanism of action of a translational repressor protein that is encoded by the regA gene of bacteriophage T4 in infected Escherichia coli host. The regA protein selectively inhibits translation of a subset of phage-induced mRNAs including its own message (autogenous translational repression). In previous years we cloned the regA gene, overproduced its protein product, purified the protein, purified the protein, and characterized some of its activities in vivo and in vitro. Some of our main results include: precise transcriptional mapping of the regA gene, localization of the target for autogenous control to the translation initiation region of the mRNA, localization of regA-protein-binding sites on several regA-regulated mRNAs, discovery of a role for regA protein in transcription termination downstream of the regA gene, and discovery of an mRNA structure that enhances translation. In the work proposed here we will investigate the roles of nucleotide sequences in the leader segment of the mRNA for regA protein in control of synthesis of the protein. Several mRNA target sites for regA protein binding (including the autogenous target) are being mapped precisely by the use of in vitro manipulations by which nucleotide replacements are made in specific sequences within the ribosome-binding domains of the regA regulated mRNAs. The effects of these in vitro- generated mutations on sensitivity to regA protein are being assessed by in vivo and in vitro translation/repression assays. In addition we are examining the role of regA protein in transcription termination at "CUUCGG" hairpin structures in intercistronic regions and attempting to identify the breakdown products of the mRNAs that the regA protein represses. A pilot study is underway aimed at identifying proteins, nucleoproteins, and RNA species that interact with regA protein and influence its activity in vivo. The work in this proposal should provide us with deeper insights into the as yet poorly understood processes by which a cell controls and coordinates its overall rates of DNA, RNA, and protein synthesis.