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. 2007 Jun 11;3(5):318–327. doi: 10.7150/ijbs.3.318

Figure 2.

Figure 2

MFNLPs and RBEIII, bind USF-1 and TFIIi. (A) Schematic representation of the HIV-1 LTR. Some transcription factors landmarks (NFAT, USF-1, RBF2, NFkB, SP-1) known to interact with target sequences on the HIV-1 LTR are indicated in their relative positions. The -121 position where the MFNLPs occur is indicated, with the MFNLP above, bound by RBF2 at the MFNLP RBEIII site. The -453 and +185 sites indicate the extremes of the LTR. The +1 site indicates the start of transcription and the +98 site, the end of Tar sequences. The dashed circles represent the approximate positions of two nucleosomes (Nuc-0 and Nuc-1). (B) RBEIII sequences specifically compete with a USF EMSA. The dsNREB oligosed as targets for the respective Hela nuclear extracts. Inclusion of competitor DNA sequences is indicated by a plus sign. (C) A USF-1 site competes specifically with RBF2 for an RBEIII site. A p32-labelled wt RBEIII ds oligo was used as target in EMSA with nuclear extracts from HeLa-s cells (NE, lanes 1-5). The target site that the respective competitor ds oligo sequences encompass, are designated with a + sign above each lane, and are listed on the right top of the panel. (D) rUSF-1 specifically binds to RBEIII, but results in a higher mobility EMSA than RBF2. A p32-labelled wt RBEIII ds oligo was used as target in EMSA with nuclear extracts from HeLa-s cells (NE, lanes 1-4) or with rUSF-1 (lanes 5-10). The target site that the respective competitor ds oligo sequences encompass, are designated with a + sign above each lane, and are listed on the right top of the panel. Lower arrow: NE-RBEIII (RBF2) complex. Upper arrow: rUSF-1-RBEIII complex. (D) rTFIIi and rUSF-1 bind cooperatively to MFNLP and wt RBEIII sites from 2 patients. A p32-labelled ds oligonucleotide target, encompassing the RBEIII site of either clone pMCE36.1 (lanes 1, 3, 5) or pMCE9.104 (lanes 2, 4, 6) was used as target in EMSA with either rUSF-1, TFIIi, or both, as indicated with a + sign above each lane, and listed at the right top of the panel. Lower arrow: Free pMCE36.1 ds oligonucleotide. Upper arrow: Free pMCE9.104 ds oligonucleotide. (B to E) The NRE, RBEIII, USF, NFκB sequences and EMSA conditions have been previously described 55, 64, 65, 67, 69.