This study has analysed some of the mechanisms involved in the transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) by transcriptional modulator proteins of the NF-ĸB/rel family. Initial attempts to purify a single NF-ĸB (p50-p65) heterodimer species from HeLa cells suggested that a family of proteins might contribute to ĸB motif DNA binding activity. HeLa cell ĸB motif DNA binding proteins were shown to be modified by glycosylation. Using circularly permuted DNA probes carrying a KB motif, it was shown that ĸB binding proteins induced significant DNA bending upon binding, while studies of the effects of the poly amine spermidine on purified HeLa cell ĸB motif DNA binding proteins showed it greatly stimulated their DNA binding activity. The DNA binding activity of native ĸB motif DNA binding proteins was also greatly stimulated by the reducing agent dithiothreitol. Using a cDNA encoding the p105 precursor to the NF-ĸB p50 subunit, the wild type DNA binding and dimerisation region (aa35-381) of p50, and three cysteine to serine mutants at cysteine residues (aa62, 119, and 273) conserved throughout the NF-ĸB/rel/dorsal family, were expressed in bacteria. The dissociation constant of the aa62 p50 mutant for the ĸB motif was 10-fold higher than that of the wild type p50. Also, dissociation rate constants for the aa62 mutant-KB motif complex in both the presence and absence of spermidine were anomalously high. The above changes suggested a different DNA binding specificity for the aa62 mutant - this was confirmed by oligonucleotide competition studies. Oligonucleotide protection experiments suggested the presence of a cysteine residue in the p50 DNA binding site-substrate protection experiments showed that this was cysteine 62, and that this residue is involved in redox regulation of p50 DNA binding activity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:750799 |
| Date | January 1993 |
| Creators | Matthews, James Robert |
| Contributors | Hay, R. T. |
| Publisher | University of St Andrews |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10023/13981 |
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