Binding of the pseudorabies virus immediate-early protein to single-stranded DNA.
In an attempt to correlate the ability to activate transcription with affinity for single-stranded DNA, both wild-type and temperature-sensitive pseudorabies virus immediate-early proteins were tested for the ability to bind to single-stranded DNA columns. Wild-type and temperature-sensitive immediate-early proteins bound to nonspecific single-stranded DNA columns with similar affinities at both 0 and 40 degrees C. There did not seem to be a direct correlation between the ability to activate transcription and the ability to bind to single-stranded DNA. To study further the interactions that are involved in binding to single-stranded DNA, we expressed the immediate-early protein in an Escherichia coli expression vector. In this system the expressed immediate-early protein was not phosphorylated, nor could it be complexed with mammalian cell factors. The first trp construct did not express a soluble form of the immediate-early protein, presumably due to the insoluble nature of the trp leader. We deleted a large segment of the trpE gene and found that the immediate-early fusion protein was soluble. We tested this protein for its affinity for single-stranded DNA by passage over single-stranded DNA cellulose columns. The bacterially expressed immediate-early protein bound single-stranded DNA at least as well as did the wild-type protein. Affinity for single-stranded DNA did not appear to be dependent on the phosphorylation state nor on the presence of mammalian cell factors.
D002846:Chromatography, Affinity; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D011558:Herpesvirus 1, Suid / Q000378:metabolism*; D017874:Immediate-Early Proteins; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C047583:pseudorabies virus immediate early proteins