Molecular genetic analysis of a prokaryotic transcriptional coactivator: functional domains of the bacteriophage T4 gene 33 protein

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Abstract
The bacteriophage T4 gene 33 encodes a small, acidic RNA polymerase-binding protein that mediates enhancement of transcriptional initiation at T4 late promoters by the T4 DNA replication accessory proteins. A set of nested deletions in the gene 33 open reading frame was constructed by oligonucleotide site-directed mutagenesis. The resulting variant gene 33 proteins were radiolabeled during overexpression employing a T7 RNA polymerase-based system and substantially purified. Each variant was analyzed for three properties of gp33: RNA polymerase binding activity, ability to mediate enhancer-dependent transcriptional activation, and repression of unenhanced transcription. Two separate regions of gp33 were required to form stable complexes with RNA polymerase, whereas the extreme carboxyl terminus of gp33 was essential for mediating late gene activation. Variant gene 33 proteins lacking the carboxyl terminus nevertheless repressed nonenhanced transcription, demonstrating that the functional domains required for transcriptional activation and repression of unenhanced transcription are separable. The possible roles of gp33 in mediating late gene expression are discussed in the light of the identification of these functional domains.