PMID:12426390

Citation	Calles, B, Salas, M and Rojo, F (2002) The phi29 transcriptional regulator contacts the nucleoid protein p6 to organize a repression complex. EMBO J. 21:6185-94
Abstract	The nucleoid protein p6 of Bacillus subtilis phage phi29 binds to DNA, recognizing a structural feature rather than a specific sequence. Upon binding to the viral DNA ends, p6 generates an extended nucleoprotein complex that activates the initiation of phi29 DNA replication. Protein p6 also participates in transcription regulation, repressing the early C2 promoter and assisting the viral regulatory protein p4 in controlling the switch from early to late transcription. Proteins p6 and p4 bind cooperatively to an approximately 200 bp DNA region located between the late A3 and the early A2c promoters, generating an extended nucleoprotein complex that helps to repress the early A2c promoter and to activate the late A3 promoter. We show that stable assembly of this complex requires interaction between protein p6 and the C-terminus of protein p4. Therefore, at this DNA region, stable polymerization of protein p6 relies on p4-specified signals in addition to the structural features of the DNA. Protein p4 would define the phase and boundaries of the p6-DNA complex.
Links	PubMed PMC137212
Keywords	Bacillus Phages/genetics; Bacillus Phages/physiology; Bacillus subtilis/virology; Bacterial Proteins/metabolism; Base Sequence; DNA Replication; DNA, Viral/genetics; DNA, Viral/metabolism; DNA-Directed RNA Polymerases/metabolism; Macromolecular Substances; Molecular Sequence Data; Promoter Regions, Genetic; Protein Binding; Protein Interaction Mapping; Repressor Proteins/chemistry; Repressor Proteins/physiology; Sigma Factor/metabolism; Structure-Activity Relationship; Transcription Factors/chemistry; Transcription Factors/physiology; Transcription, Genetic; Viral Proteins/chemistry; Viral Proteins/physiology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPPH2:NP		GO:0006351: transcription, DNA-templated	ECO:0000315:		P		The wildtype gel in figure 1 supports transcriptional activity under normal conditions, and it is contrasted and supported by the change in transcription activity recorded in neighboring gels.	complete CACAO 12442
BPPH2:NP		GO:0017053: transcriptional repressor complex	ECO:0000315:		C		Figure 2B. A DNase I footprinting assay with both the wild type and mutant was run to observe the p6 and p4 complexes with a DNA fragment containing promoters A3, A2b, and A2c. Calles et al. incubated each lane with an increasing concentration of p6 or p4 and observed the footprints, or areas where the DNase fails to cut because of complex bounded onto the DNA. Mutant p4 proteins lead to a decrease in repression due to the failure of the p4-p6 nucleocomplex protein to form, showing the importance of the p4 protein in transcriptional repression. The presence of p4 protein increases the affinity of the p6 protein to bind, which would repress transcription.	complete CACAO 12503
BPPH2:NP		GO:0017053: transcriptional repressor complex	ECO:0000314:		C		Figure 3. Competitive EMSA with increasing amounts of [p4] and [p6] incubated in each lane to observe the p4-p6 nucleoprotein complex formation at the late A3 promoter between positions -58 to -104. P4 by itself binds downstream of -58 position; P6 by itself binds throughout the -58 to -104 position; suggesting that the p4 protein allows specific sites for the complex to form.	complete CACAO 12666
BPPH2:NP		GO:0001217: bacterial-type RNA polymerase transcriptional repressor activity, sequence-specific DNA binding	ECO:0000315:		F		In figure 4, RNAP binding to the promoters in phage phi 29 is monitored in the presence of proteins p4 and p6 with or without mutations at the C-end. The gel shows the inability of a stable p4-p6 complex to form in order to properly repress transcription (ie. blocking the binding of RNAP to the A2c promoter), due to a mutation at the C-end of the p4 protein.	complete CACAO 12841

References

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