A DNA CURVATURE CAN SUBSTITUTE PHAGE-PHI-29 REGULATORY PROTEIN-P4 WHEN ACTING AS A TRANSCRIPTIONAL REPRESSOR

Binding of phage phi-29 regulatory protein p4 to its target sequences produces a strong bend in the DNA that is important for activation of the late A3 promoter (P(A3)). Protein p4 binding site in P(A3) overlaps with the divergently transcribed main early promoter, P(A2b), which suggested that p4 could also act as a repressor. We show that protein p4 both excludes Bacillus subtilis sigma-A-RNA polymerase from P(A2b) and directs it to the divergently transcribed A3 promoter. Although steric hindrance is likely to be involved in the repression process, we have also analysed the effect on P(A2b) activity of a sequence-dependent curvature that simulates that induced by protein p4. A progressive increase in the DNA curvature of protein p4 binding region, performed by site-directed mutagenesis, has indicated that a static DNA curvature by itself can inhibit transcription from P(A2b), both by impairing the binding of sigma-A-RNA polymerase to the promoter and by reducing its ability to form transcriptionally active open complexes. These results indicate that bending promoter sequences in a direction unfavourable for RNA polymerase binding can repress transcription. Protein p4-induced DNA bending could therefore participate in P(A2b) repression by producing a DNA structure not recognized as a promoter by sigma-A-RNA polymerase.