Transcriptional activation by the tyrosine repressor protein

Qing Bai, Purdue University

Abstract

The TyrR protein of Escherichia coli is a dimeric σ 70-specific transcription factor containing 513 amino acid residues per subunit. It is of particular interest for several reasons. First, it bears a high degree of structural identity to a large and functionally diverse group of prokaryotic transcription factors (the NtrC superfamily), most of which are specific for σ54 promoters. Second, it can either activate or repress transcription, depending on the target promoter. Third, it has the ability to bind ATP, hydrolyze ATP, self- phosphorylate, and self-dephosphorylate. How these catalytic activities affect its transcription factor activity is unknown. The role of TyrR as an activator of the tpl promoter of Citrobacter freundii was analyzed. Three TyrR binding sites, designated Boxes A, B, and C, are centered at coordinates −272.5, −158.5, and −49.5, with respect to the transcriptional startpoint. TyrR binds cooperatively to these sites. Full activation of tpl required two additional proteins, integration host factor (IHF) and the cyclic AMP receptor protein (CRP). The binding sites for IHF and CRP were identified by DNase I footprinting. The proposed roles of these accessory proteins are to introduce bends into tpl promoter DNA between Boxes A, B and C so as to facilitate the formation of higher order complexes between DNA-bound TyrR proteins, thereby satisfying a condition for activation of the tpl promoter. The tyrR genes of C. freundii and Salmonella typhimurium were cloned and structurally characterized. TyrR proteins of 514 and 513 amino acid residues with 80% identity to the TyrR protein of E. coli are predicted from the DNA sequences. All three TyrR proteins, when compared to their σ54 counterparts, have a unique gap of eight residues at sequence positions 282/283. A number of deletion and insertion mutations were constructed that involved the TyrR protein of C. freundii and a related σ54 transcription factor, PhhR of Pseudomonas aeruginosa. The insertion of the missing residues into the TyrR protein of E. coli did not affect the ability to bind operator DNA. However, the resulting mutant protein did not appear to acquire activation capability for a synthetic σ 54 promoter. The TrpH protein of E. coli was overexpressed following the induction of T7 RNA polymerase in cells harboring a plasmid where the trpH gene was under the control of the phage T7 promoter. In E. coli, the transcriptional startpoint for trpH mRNA, characterized by primer extension, is separated from the startpoint of the trp operon by 162 bp of DNA. Upstream of the trpH promoter there is a weak binding site for the TrpR protein, centered at coordinate −63.5 with respect to the trpH transcriptional startpoint. Under certain experimental conditions, the TrpR protein stimulates the expression of the trpH gene. The phenotype of a null mutant suggests that the TrpH protein can oppose the termination of transcription at the trp attenuator. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Somerville, Purdue University.

Subject Area

Molecular biology

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