Gene expression of bacteriophage ø29 is carried out by the Bacillus subtilis RNA polymerase and is regulated by early viral proteins. The switch from early to late transcription involves the formation of an extended nucleoprotein complex of proteins p4 and p6 in the 219 bp sequence of DNA comprising A2c, A2b and A3 promoters. In this work we analyzed the determinants of the specificity of p4-DNA interaction, the role of the p4 binding sites and the regulation of transcription in a ø29-related phage. Early transcription produces proteins implicated mainly in the synthesis of ø29 DNA and transcription regulators; late transcription gives rise to structural proteins required for production of the infective viral particles. Protein p6 is a nucleoid protein, small, dimeric, and very abundant in infected cells; nucleoid proteins bind DNA non-specifically, although some of them show a preference for certain sequences or structures of DNA. Protein p4 is the phage transcriptional factor. It binds to the DNA at two regions. Region 1 is located between early promoters A2c and A2b and region 2 is located between promoter A2b and the late promoter A3. Each region has two binding sites; p4 binds as a dimer to each binding site. By means of directed mutagenesis of different residues of the protein and by modification of nucleotides at the binding sites we could define the principal characteristics of a p4 binding site and the determinants of the specificity on the p4- DNA complex; we could observe, moreover, that p4 does not present the same affinity either for its binding sites or for the sequence at each end of the binding site. These results lead us to propose a zipper mechanism for the binding of p4. Here we demonstrate that p4 does not require an intrinsically bent DNA for binding and that whenever p4 binds to a complete region, it bends the DNA and induces a curvature around 86 º. Studies of the role of each binding site allowed us to conclude that sites 2 and 4 do not have a direct implication in the regulation of the switch from early to late transcription. The functional complex is formed by binding of p4 at sites 1 and 3 and the sequence comprised between them is covered by protein p6, being this complex able to repress early transcription and simultaneously activate the late transcription. This mechanism is conserved in another B. subtilis phage, Nf, a phage from a different group of the ø29 family. Nf bacteriophage encodes proteins p4N and p6N, homologous to ø29 early proteins p4 and p6, respectively, and contains two early promoters homologous to A2c and A2b and a late promoter similar to A3. Transcriptional switch also requires the formation of a p4-p6 nucleoprotein complex. However, ø29 proteins are unable to regulate the Nf promoters, suggesting that the transcriptional regulation mechanism of each phage has evolved differently.

El trabajo presentado en esta Tesis Doctoral ha sido realizado en el Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM) bajo la dirección de la Dra. Ana Camacho Pedrero. Para la realización de este trabajo se contó con una beca de Formación de Personal Investigador de la Comunidad Autónoma de Madrid.

Tesis doctoral inédita. Universidad Autónoma de Madrid. Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-02-2007

Peer reviewed