A GC-box motif upstream of the B19 parvovirus unique promoter is important for in vitro transcription

Abstract

Nucleotides upstream of the B19 parvovirus P6 promoter affect in vitro transcription in HeLa cell nuclear extracts. Comparison of the relative transcriptional strengths of equimolar mixes of plasmids containing the intact upstream sequence and plasmids containing deletions within these nucleotides identified several regions that affect transcription in vitro. A fragment containing two of five GC-box motifs which correspond to high-affinity SP1-binding sites was shown, by using a gel shift assay, to bind a HeLa cell factor (or factors). DNase I, methylation interference, and methylation protection footprinting demonstrated that the HeLa cell factor(s) bound to one of the two GC-box motifs within this fragment. Mutation of this GC box abolished factor binding and significantly reduces in vitro transcription from the P6 promoter. These results suggest that the B19 parvovirus promoter includes a complex regulatory region containing multiple sequences which affect promoter strength and that the GC-box motif is a major controlling sequence for in vitro transcription.