Repression of Bacteriophage φ29 Early Promoter C2 by Viral Protein p6 Is Due to Impairment of Closed Complex

Ana Camacho,Margarita Salas

doi:10.1074/jbc.m103738200

Abstract

Bacillus subtilis phage phi 29 encodes a very abundant protein, p6, which is a non sequence-specific DNA-binding protein. Protein p6 has the potential to bind cooperatively to the phage genome, forming a nucleoprotein complex in which the DNA adopts a right-handed toroidal conformation winding around a protein core. The formation of this complex at the right end of the phage genome where the early promoter C2 is located affects local topology, which may contribute to the promoter repression, although the underlying molecular mechanism of this repression is not presently known. In this study, we analyzed the effect of the p6 nucleoprotein complex on the formation of transcription complexes at the C2 promoter. The results obtained indicate that the nucleoprotein complex does not occlude promoter C2 to RNA polymerase because both proteins can bind to the same DNA molecule. Protein p6 binds along the fragment including the sequence adjacent to the bound polymerase, altering the structure of the transcriptional complex and affecting specifically the stability of the closed complex. The findings presented might help to answer some of the open questions about the concerted molecular mechanisms of histone-like proteins as transcriptional silencers.

Highlights

Bacteriophage ␾29 gene expression is regulated at the transcription initiation step either by repressing strong early promoters or by activating the otherwise poorly expressed late promoter (Ref. 1 and see Fig. 1)
The results obtained indicate that the nucleoprotein complex does not occlude promoter C2 to RNA polymerase because both proteins can bind to the same DNA molecule
Nothing is known about the molecular mechanism by which p6 represses promoter C2, several alternative, not mutually exclusive, models can be envisioned to explain the effect of p6 on promoter C2 activity: (i) the binding of p6 to the genome right end might occlude the promoter in the nucleoprotein complex; (ii) the p6-induced modification of promoter architecture might impair the RNA polymerase (RNAP) bending needed for the formation of a stable transcription complex; (iii) protein p6 bound at the upstream sequence of the promoter might help to block transcription by impeding a postbinding step

Summary

Introduction

Bacteriophage ␾29 gene expression is regulated at the transcription initiation step either by repressing strong early promoters or by activating the otherwise poorly expressed late promoter (Ref. 1 and see Fig. 1). The p61⁄7nucleoprotein complexes formed at the genome ends are involved in different biological functions, such as activation of the initiation of DNA replication and repression of the transcription derived from promoter C2 [5, 6]. Nothing is known about the molecular mechanism by which p6 represses promoter C2, several alternative, not mutually exclusive, models can be envisioned to explain the effect of p6 on promoter C2 activity: (i) the binding of p6 to the genome right end might occlude the promoter in the nucleoprotein complex; (ii) the p6-induced modification of promoter architecture might impair the RNAP bending needed for the formation of a stable transcription complex; (iii) protein p6 bound at the upstream sequence of the promoter might help to block transcription by impeding a postbinding step. We show that the p61⁄7DNA nucleoprotein complex does not seem to occlude the C2 promoter sequence to the RNAP but rather affects the stability of the closed complex

Objectives

Results

Conclusion