Oligomeric structures of the phage phi29 histone-like protein p6.

Protein p6 of Bacillus subtilis phage phi29 has been described as a histone-like protein, playing a role in genome organization and compaction, on the basis of its high intracellular abundance, its pleiotropic effect, and its ability to bind and highly compact the whole phi29 DNA in vitro. Protein p6 forms large multimeric nucleoprotein complexes in which a right-handed superhelical DNA wraps toroidally around the protein core. Analytical ultracentrifugation analysis, at the concentration estimated in vivo (at least 1 mM), showed that protein p6 self-associates into elongated oligomers, suggesting that, in the absence of DNA, the protein could form a scaffold for DNA binding. In this work we have studied the structure of these oligomers by transmission electron microscopy and image processing. The results show that protein p6 aggregates into crooked-shaped oligomers, compatible with a helical structure. The oligomers could interact head-to-tail to form doughnut-shaped structures or they could grow into right-handed double-helical filaments by a nucleation-dependent polymerization process. The dimensions of the crooked-shaped structures are in agreement with that of the DNA in the nucleoprotein complex previously described. We propose that the crooked-shaped structures could act as a scaffold imposing the right-handed path followed by the DNA, and thus it could be considered a non-transient DNA chaperone.     

Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication.     

Analytical Ultracentrifugation Studies of Phage ?29 Protein p6 Binding to DNA

Protein p6 from Bacillus subtilis phage ?29 binds double-stranded DNA, forming a large nucleoprotein complex all along the viral genome, and has been proposed to be an architectural protein with a global role in genome organization. Here, we have characterized quantitatively the DNA binding properties of protein p6 by means of sedimentation velocity and sedimentation equilibrium experiments permitting determination of the strength and stoichiometry of complex formation. The composition dependence of protein binding to DNA is quantitatively consistent with a model in which the protein undergoes a reversible monomer-dimer self-association, and the dimeric species binds noncooperatively to the DNA. We also have found that when the anisotropic bendability periodicity of the nucleotide sequence preferred by p6 is modified, nucleocomplex formation is impaired. In addition, suppression of complex formation at high ionic strength is reversed by the addition of high concentrations of an inert polymer, mimicking the crowded intracellular environment. The results obtained in this work illustrate how macromolecular crowding could act as a metabolic buffer that can significantly extend the range of intracellular conditions under which a specific reaction may occur.     

Overproduction and purification of protein P6 of Bacillus subtilis phage phi 29: role in the initiation of DNA replication.

A phi 29 DNA fragment containing gene 6, required for DNA replication, has been cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda. A polypeptide with an electrophoretic mobility close to that of p6 was labelled with 35S-methionine after heat induction. This protein, representing about 4% of the total E. coli protein after 1 h of induction, was obtained in a highly purified form. The protein was characterized as p6 by amino acid analysis and NH2-and COOH-terminal sequence determination. Protein p6 has an apparent molecular weight of 23,600, suggesting that the native form of the protein is a dimer. The purified protein p6 stimulated the protein-primed initiation of phi 29 DNA replication when added to purified proteins p2 (phi 29-coded DNA polymerase) and p3 (terminal protein).     

The cyclic AMP (cAMP)-cAMP receptor protein complex functions both as an activator and as a corepressor at the tsx-p2 promoter of Escherichia coli K-12.

The tsx-p2 promoter is one of at least seven Escherichia coli promoters that are activated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and negatively regulated by the CytR repressor. DNase I footprinting assays were used to study the interactions of these regulatory proteins with the tsx-p2 promoter region and to characterize tsx-p2 regulatory mutants exhibiting an altered response to CytR. We show that the cAMP-CRP activator complex recognizes two sites in tsx-p2 that are separated by 33 bp: a high-affinity site (CRP-1) overlaps the -35 region, and a low-affinity site (CRP-2) is centered around position -74 bp. The CytR repressor protects a DNA segment that is located between the two CRP sites and partially overlaps the CRP-1 target. In combination, the cAMP-CRP and CytR proteins bind cooperatively to tsx-p2, and the nucleoprotein complex formed covers a region of 78 bp extending from the CRP-2 site close to the -10 region. The inducer for the CytR repressor, cytidine, does not prevent in vitro DNA binding of CytR, but releases the repressor from the nucleoprotein complex and leaves the cAMP-CRP activator bound to its two DNA targets. Thus, cytidine interferes with the cooperative DNA binding of cAMP-CRP and CytR to tsx-p2. We characterized four tsx-p2 mutants exhibiting a reduced response to CytR; three carried mutations in the CRP-2 site, and one carried a mutation in the region between CRP-1 and the -10 sequence. Formation of the cAMP-CRP-CytR DNA nucleoprotein complex in vitro was perturbed in each mutant. These data indicate that the CytR repressor relies on the presence of the cAMP-CRP activator complex to regulate tsx-p2 promoter activity and that the formation of an active repression complex requires the combined interactions of cAMP-CRP and CytR at tsx-p2.     

Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex.