Transcription activation at a distance by phage phi 29 protein p4. Effect of bent and non-bent intervening DNA sequences.
Protein p4 of the Bacillus subtilis phage phi 29 switches on the transcription of the viral late genes by binding to the viral late promoter at a region close to the RNA polymerase binding site. Gel retardation and DNase I footprinting assays show that the presence of protein p4 is required for RNA polymerase recognition of the late promoter. The protein p4 and RNA polymerase DNA binding sites have been separated by the insertion of bent and non-bent DNA sequences of different lengths. These mutant promoters were used to study in vitro their protein p4-dependent transcriptional activity and their interaction with both protein p4 and RNA polymerase. The results indicate that protein p4 is able to function at longer DNA distances from the RNA polymerase binding site than in the natural promoter. The extent of protein p4 activity depended on the length and conformation of the inserted DNA. Activation of transcription and RNA polymerase binding was favoured when the relative orientation of protein p4 and RNA polymerase was conserved and when the intervening DNA had a bent conformation. These data, together with the DNase I footprints, suggest that activation at distance by protein p4 involves a DNA loop held by the interaction of protein p4 and RNA polymerase.
Serrano
M
M
Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Barthelemy
I
I
Salas
M
M
D001412:Bacillus subtilis / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D007438:Introns; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D014157:Transcription Factors; D014764:Viral Proteins; C053987:p4 protein, Bacteriophage phi 29; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I