Bacteriophage T4 RegA protein binds to the Shine-Dalgarno region of gene 44 mRNA

Abstract

We have overproduced and purified wild type regA protein, a translational repressor encoded by bacteriophage T4. The repressor activity of the cloned regA protein has been tested on four known regA target genes (T4 genes: 44, 45, rpbA and regA) using in vitro coupled transcription-translation reactions. We have demonstrated the sensitivity of two additional T4 genes coding for α-and β glucosyltransferases to regA protein in vitro. The regA target site on the gene 44 messenger RNA has been identified through deletion analysis and RNase protection assays, using plasmids containing gene 44-lacZ fusions. The effect of regA protein on expression of 44P-β-galactosidase fusion proteins was assayed in vitro in coupled transcription-translation reactions. Analysis of deletion mutants of gene 44-lacZ localized the regA recognition region to between nucleotides −11 and +9 of the mRNA. RNase protection assays of g44-lacZ transcripts further defined the site of regA protein interaction to between nucleotides −10 and +2 of the mRNA. This region overlaps the gene 44 Shine-Dalgarno region and the A and U of the initiation codon.