Purification of RBF-2, a transcription factor with specificity for the most conserved cis-element of naturally occurring HIV-1 LTRs.
The combination of high turnover and error-prone reverse transcription results in naturally occurring human immunodeficiency virus-1 long terminal repeats that differ considerably from the prototype sequence. Although no transcription-factor-binding site escapes mutation, the only mutated site that appears to be invariably compensated by co-occurrence of its duplication is the RBE III site, a target for the transcription factor RBF-2. In this work, we characterize RBF-2 further by biochemical purification. RBF-2 was purified by chromatography on heparin agarose and Mono-Q ion exchange chromatography, followed by affinity chromatography on mutant and wild-type RBE III oligonucleotide columns. The purified RBF-2 preparation contained 4 major and 1 minor polypeptides of 50, 100, 110, 120 and 125 kD, as detected by silver staining in SDS-PAGE gels. UV cross-linking revealed a specific 100-kD species, indicating that this protein likely represents the DNA-binding component of a complex. A second factor with DNA-binding specificity similar to that of RBF-2, called RBF-B, was also identified by heparin-agarose fractionation, which suggests that effects of the RBE III cis-element may be mediated by a combination of factors in vivo.
Estable
M C
MC
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, B.C., Canada.
Hirst
M
M
Bell
B
B
O'Shaughnessy
M V
MV
Sadowski
I
I
D000837:Anion Exchange Resins; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid; D017124:Conserved Sequence / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D019169:Jurkat Cells; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012117:Resins, Synthetic; D020218:Response Elements / Q000235:genetics; D012685:Sepharose / Q000031:analogs & derivatives; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D000837:Anion Exchange Resins; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D012117:Resins, Synthetic; D014157:Transcription Factors; C037405:heparin-sepharose; C042855:Mono Q; D012685:Sepharose