Figure 1B shows, as expected, that a minimal rabbit promoter was virtually inactive, whereas transcriptional activity was greatly increased by addition of the SV40 enhancer. Thus, in mammalian cells transcription of protein-coding genes entails option TCs that differ by the presence or absence of TAFs. Introduction In eukaryotes, gene regulation is largely controlled at the transcriptional level. Factors involved in the accurate transcription of eukaryotic structural genes by RNA polymerase II (class II genes) can be classified into two groups. First, general (or basic) transcription factors (GTFs) are necessary and can be sufficient for accurate transcription initiation in vitro (for evaluate, observe [1]). Such factors include RNA polymerase II itself and at least six GTFs: TFIID, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH. The GTFs assemble around the core promoter in an ordered fashion to Ras-GRF2 form a transcription pre-initiation complex (PIC). The first step in PIC assembly is binding of the GTF TFIID to the TATA box. TFIID is R112 usually a multi-subunit complex consisting of the TATA-box-binding protein (TBP) and a set of tightly bound TBP-associated factors (TAFs) [2,3,4]. Transcriptional activity is usually greatly stimulated by the second class of factors, promoter-specific activator proteins (activators). In general, cellular activators are sequence-specific DNA-binding proteins whose acknowledgement sites are usually present in sequences upstream of the core promoter (examined in [5,6]). In addition to a sequence-specific DNA-binding domain name, a typical activator also contains a separable activation domain name. A variety of studies show that activators work, at least in part, by increasing PIC formation through a mechanism thought to involve direct interactions with one or more components of the transcription machinery [1,7,8,9]. Activators can also take action through other mechanisms, such as increasing the rate of transcriptional elongation, promoting multiple rounds of transcription, and directing chromatin modifications (examined in [10]). The Tat protein of human immunodeficiency computer virus type I (HIV-1) is usually a potent activator of the viral long terminal repeat (LTR) and is required for viral replication (examined in [11,12,13]). Unlike common activators, which bind to promoter DNA, Tat binds to nascent viral RNA through an RNA-binding site termed the TAR element. Tat function entails direct interaction with the cellular cofactor called positive transcription elongation factor b (P-TEFb), which is composed of two subunits, cyclin T1 (CycT1) and CDK9 (examined in [14,15]). Several lines of evidence have suggested that unlike common activators, Tat stimulates transcriptional R112 elongation rather than initiation. According to the current model (examined in [11,12,13]), in the absence of Tat, RNA polymerase II initiates from your HIV-1 LTR in a form unable to elongate efficiently and thus stalls (or pauses) near the transcription start site. When present, Tat binds to TAR and recruits P-TEFb, allowing CDK9 to phosphorylate the C-terminal domain name of the RNA polymerase II large subunit, thereby increasing transcriptional processivity. The chromatin immunoprecipitation R112 (ChIP) assay has provided a powerful approach to study in vivo the mechanism by which activators stimulate transcription and to delineate the composition of transcription complexes (TCs). In yeast, ChIP analysis has been used to show that activators function, at least in part, by stimulating TC assembly. These studies have also revealed that at certain promoters TBP is usually recruited in the absence of TAFs [16,17], indicating that, at least in yeast, some TCs contain TBP but not TFIID. Here we perform ChIP experiments to study the mechanism of transcription activation by Tat in vivo. Results Experimental Design To analyze transcription activation by Tat and other activators we performed ChIP experiments in transiently transfected mammalian tissue culture cells. Plasmids expressing a reporter construct, containing a core promoter and various combinations of activator-binding sites, were.