The general transcription factors (GTFs) of eukaryotic RNA polymerase II, in a process facilitated by regulatory and accessory factors, target promoters through synergistic interactions with core elements. The specific binding of the TATA box-binding protein (TBP) to the TATA box has led to the assumption that GTFs recognize promoters directly, producing a preinitiation complex at a defined position. Using biochemical analysis as well as biophysical single-pair Förster resonance energy transfer, we now provide evidence that negative cofactor-2 (NC2) induces dynamic conformational changes in the TBP-DNA complex that allow it to escape and return to TATA-binding mode. This can lead to movement of TBP along the DNA away from TATA.

The herpes simplex virus type 1 DNA polymerase consists of a catalytic subunit (POL or UL30) and a processivity factor (UL42). The POL/UL42 interaction, which occurs through the extreme C-terminus of POL, is essential for HSV-1 replication and thus represents a valid target for drug inhibition. We recently showed (A. Loregian et al. (1999) Proc. Natl. Acad. Sci. USA 96, 5221-5226) that an oligopeptide corresponding to the 27 C-terminal amino acids of POL, when delivered into herpes simplex virus type 1-infected cells by a protein carrier, was able to localize into the nucleus and to inhibit viral replication by disruption of the POL/UL42 interaction. In this report, to further characterize the 27 mer (Pol peptide), we investigated whether its nuclear localization was due to the presence of a nuclear localization signal. By testing the ability of the Pol peptide to localize the beta-galactosidase, a normally cytoplasmic protein, to the nucleus, we confirmed that the Pol peptide contained a functional nuclear localization signal, corresponding to the RRMLHR motif. This sequence proved not only necessary but also sufficient for nuclear localization, because its substitution with a six-alanine stretch prevented nuclear translocation of the beta-galactosidase-Pol peptide fusion. Site-directed mutagenesis experiments on this revealed that both the three basic arginines and the two hydrophobic residues Met and Leu were crucial for nuclear targeting. Finally, functionally equivalent sequences were also found in the C-terminus of the catalytic subunits of human cytomegalovirus (RRLHL) and of equine herpesvirus-1 DNA polymerase (RRILH).

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2⅐TBP with DNA. We show that NC2⅐TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP⅐TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATAcontaining and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2⅐TBP displays a K D for DNA of ϳ2 ؋ 10 ؊9 M for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP⅐DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence.TATA-binding protein (TBP) 1 binds to eukaryotic promoters to nucleate initiation of transcription (1-3). The crystal structure of the TBP⅐DNA complex revealed a saddle-shaped conformation of TBP in which the highly conserved carboxyl terminus forms a concave surface that interacts with the minor groove of DNA. As a consequence, the minor groove becomes dramatically widened, and the DNA is severely bent (4). The characteristic bend as well as the TBP conformation is conserved in the co-crystal structures of TBP⅐TFIIA⅐DNA (5, 6), TBP⅐TFIIB⅐DNA (7), and TBP⅐NC2⅐DNA (8).The thermodynamics and kinetics of TBP-TATA interactions have been investigated in detail. Both yeast and human TBP show specificity for TATA boxes. Values for the K D are in the range of 5 ϫ 10 Ϫ10 to 2 ϫ 10 Ϫ8 M for yeast TBP (9 -13) and 4.6 ϫ 10 Ϫ10 to 1 ϫ 10 Ϫ9 M for human TBP (14, 15). Variations may be accounted for in part by the specific conditions and the different methods employed, which are gel shifts and footprints versus solution FRET studies. Specificities of TBP for TATA range from Ͻ1 order of magnitude if a canonical TATA is compared with single point mutants in the TATA sequence to roughly 3 orders of magnitude relative to random DNA (14). Human TBP has been proposed to bind DNA through the conserved domain in the usual specific manner or, alternatively, in a nonspecific manner that depends on its non-conserved amino-terminal region (16).Several factors modulate binding of TBP to DNA. The TBPassociated factors (TAFs) support binding of TBP specifically to TATA-less promoters via DNA contacts through cognate promoter sequences (17, 18). The general transcription factor TFIIA accelerates and stabilizes binding of TBP to TATA boxes (19 -21). It also functions as a co-activator for some transcriptional activators (22-26). TFIIA further functions as an antagonist of NC2 (also called Dr1/DRAP) (27, 28). Evidence for this equilibrium between NC2 and TFIIA stems from both biochemical investigations in human cells and genetic studies in yeast in which mutants in TFIIA genes w...

The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2␣ and NC2␤, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2␣ and NC2␤ depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin ␣/␤ heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as cotransfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2␤. In contrast, NC2␣ does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2␣ masks the NES in NC2␤, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin ␣/␤-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits. The negative cofactor 2 (NC2)2 is a protein complex composed of two subunits, NC2␣ (DRAP1) and NC2␤ (Dr1). Both subunits are conserved in eukaryotes and essential for Saccharomyces cerevisae viability (1, 2). NC2␣ and NC2␤ heterodimerize via histone-fold domains and associate with the promotor-bound TATA-binding protein (3, 4). The resulting NC2-TATA-binding protein-DNA complex sterically hinders the recruitment of transcription factor IIB and in part of transcription factor IIA (5), and thus inhibits transcription initiation (6, 7). The NC2 complex is present on a substantial fraction of human genes (8). Besides mediating TATA-binding protein binding to TATA-containing and TATA-less promoters (9) the NC2 complex can also mobilize TATA-binding protein on the DNA (10). In addition to the well established function as transcriptional repressor (11) several studies have shown that NC2 activates transcription, in vitro and in vivo (12-16). The mechanism underlying the positive effects of NC2 on gene expression is not understood. Although the two NC2 subunits mostly function together, recent studies in S. cerevisae (17), Drosophila (18), and human provided evidence that NC2␣ and NC2␤ can associate with different proteins (19 -21). In this study, we have analyzed whether human NC2 subunits contain localization signals that permit individual crossing of the nuclear membrane or whether nuclear import of NC2␣ and NC2␤ depends on heterodimerization.During interphase the exclusive site of nucleocytoplasmic exchange is the nuclear pore complex...