Purification of the DNA binding domain of herpes simplex virus type 1 immediate-early protein Vmw175 as a homodimer and extensive mutagenesis of its DNA recognition site.
The herpes simplex virus type 1 (HSV-1) Immediate-Early (IE) polypeptide Vmw175 is essential for the activation of transcription from viral early and late promoters. Vmw175 also reduces the activity of its own (IE-3) promoter in transfection assays. Both transactivation and repression mediated by Vmw175 require the integrity of a conserved domain of the polypeptide which has been shown to bind to specific DNA sequences. We have investigated the DNA sequence requirements for Vmw175 binding using a randomly mutated target. The results indicate that the binding site covers a region of 13 nucleotides divided into proximal and distal parts which are consistent with the consensus ATCGTNNNNNYSG. We have also expressed several different constructs encompassing the DNA binding domain of Vmw175 in bacteria, and obtained preparations of greater than 90% purity. The DNA binding domain is a dimer in solution, and binds DNA with a specificity similar to that of the intact protein, although the smallest DNA binding competent protein has a slightly reduced specificity.
Everett
R D
RD
Medical Research Council Virology Unit, Glasgow, UK.
Elliott
M
M
Hope
G
G
Orr
A
A
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D018139:Simplexvirus; D013329:Structure-Activity Relationship; D013604:T-Phages / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D015534:Trans-Activators; D004247:DNA