Purification of RBF-2, a transcription factor with specificity for the most conservedcis-element of naturally occurring HIV-1 LTRs

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Abstract
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 IIIcis-element may be mediated by a combination of factors in vivo.