The DNA‐binding properties of an artificial 42‐residue polypeptide derived from a natural repressor

Abstract

Bacteriophage 434 repressor recognizes the operator sequences ACAAG and ACAAT. As the same or similar sequences occur in the enhancer region of HIV-l, 434 repressor was a potential HIV enhancer-binding protein. We found that the interaction of the DNA-binding domain of 434 repressor with a 57-bp HIV enhancer DNA was very weak whereas a 42-residue construct, comprising the recognition helix and four copies of a positively charged segment of the repressor, bound strongly. The results of footprint and cell-free in vitro transcription studies showed that the 42-residue peptide bound preferably to the enhancer region of HIV-l and acted as an artificial repressor. Replacement of an essential glutamine of the recognition helix by glutamic acid resulted in a partial shift of the sequence specificity of the 42-residue peptide.

Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells

“…In summary, based on previous work [14, 15, 18, 26], we have designed a 64-residue peptide (R64, Figure 1(b)) containing a central HIV-1 enhancer recognition helix and terminal nuclear localization and plasma membrane transduction signals. This peptide bound to the two HIV-1 enhancers and thus competed with NF- κ B, the major activator of HIV-1 replication [46, 47].…”

“…The recognition helix of 434 repressor was used because the nucleotide sequences of the two enhancers in the 5′-LTR region of HIV-1 were identical to repressor-binding sequences of the 434 operators, and shorter versions of R64 lacking Arg 9 [14] or both the Arg 9 and the NLS sequences (R42, [14, 15]) had been shown to bind to the enhancer region of synthetic HIV-1 LTR DNA, thereby displacing the p50 subunit of NF- κ B from its two binding sites in LTR and inhibiting HIV-1 enhancer-driven transcription [14, 15]. …”

Fusion Proteins from Artificial and Natural Structural Modules

“…The enhancer region (positions -104 to -80) contains two GGGACTTTCC NF-κB binding sites. c) Sequence of R42, the artificial 42-residue 434 repressor-derived HIV-1 enhancerbinding peptide [37]. The recognition helix of 434 repressor is underlined.…”

“…6c) [36,37]. This peptide bound tightly to the HIV-1 enhancer region, as shown by band shift assays in the presence of a 2500-fold molar excess of competitor DNA [38], stepwise displacement of the p50 subunit of NF-κB from its two binding sites [38] and DNase I footprinting [38], and inhibited the cell-free in vitro transcription of HIV-1 enhancer-containing plasmids [37,39]. All R42 mutants prepared so far including those in which the recognition helix had been replaced by the entire helix-turn-helix motif of the DNA-binding domain of bacteriophage 434 repressor [38], had inferior HIV-1 enhancer binding properties compared with those of R42.…”

“…Binding, however, became much stronger and remarkably specific after two copies each of the repressor sequence 37-44 (Gly Lys Thr Lys Arg Pro Arg Phe) had been added to the NH 2 -terminus and the COOH-terminus of the recognition helix of the repressor (residues 28-36, Gln Gln Ser Ile Glu Gln Leu Glu Asn) resulting in a 42-residue peptide (R42, Fig. 6c) [36,37]. This peptide bound tightly to the HIV-1 enhancer region, as shown by band shift assays in the presence of a 2500-fold molar excess of competitor DNA [38], stepwise displacement of the p50 subunit of NF-κB from its two binding sites [38] and DNase I footprinting [38], and inhibited the cell-free in vitro transcription of HIV-1 enhancer-containing plasmids [37,39].…”