Nucleotide-sequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA

The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding pr otein that represses translation of its own mRNA. Previous studies implicat ed two segments of the untranslated 5'-leader of the mRNA in repressor bind ing, an RNA hairpin structure and the adjacent RNA to the 3' side, which co ntains the Shine-Dalgarno sequence. Here, we show by in vitro gp43-RNA bind ing assays that both translated and untranslated segments of the mRNA contr ibute to the high affinity of gp43 to its mRNA target (translational operat or), but that a Shine-Dalgarno sequence is not required for specificity. Nu cleotide sequence specificity appears to reside solely in the operator's ha irpin structure, which lies outside the putative ribosome-binding site of t he mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restrict ed arrangement of purine relative to pyrimidine residues and an invariant 5 '-AC-3' in the unpaired (loop) segment of the RNA structure. We propose a g eneralized structure for the hairpin that encompasses these features and di scuss possible relationships between RNA binding determinants of gp43 and D NA binding by this replication enzyme.