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Open Access item Especificidad en la interacción de la proteína p4 con el DNA y formación del complejo regulador de la transcripción del bacteriófago phi29
|Authors:||Pérez García, Laura|
|Keywords:||Bacteriófago phi29, Replicación de ADN|
|Publisher:||Universidad Autónoma de Madrid|
|Abstract:||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.|
|Description:||Tesis doctoral inédita. Universidad Autónoma de Madrid. Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-02-2007|
|Appears in Collections:||(CBM) Tesis|
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