Binding of the bacteriophage T4 regA protein to mRNA targets: an initiator AUG is required.
Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression. We have obtained large amounts of regA protein.
Unnithan
S
S
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309.
Green
L
L
Morrissey
L
L
Binkley
J
J
Singer
B
B
Karam
J
J
Gold
L
L
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013604:T-Phages / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D003062:Codon; D012333:RNA, Messenger; D012367:RNA, Viral; C066700:RegA protein, Enterobacteria phage T4; D014764:Viral Proteins