Tat associates with TFIIH in the absence of the Pol II holoenzyme in vivo. (A) Total cell lysates (TCL) from COS cells were immunoprecipitated with αCIITA (lane 2) or αERCC3 antibodies (lane 3). One-third of the lysate was used as the input control (lane 1). Samples were separated by SDS-PAGE, transferred to membranes, and probed with the antibodies indicated on the left. (B) COS cells expressed HA-tagged wild-type (Tat) or mutant (C30G or K41A) Tat proteins. Cell lysates were immunoprecipitated with either the αCIITA (C) antibody or the αERCC3 antibody (αERCC3). Equal amounts of the lysates were loaded as input controls. Samples were processed as in A and probed with the αHA antibody. (C) Nuclear extracts (NE) or lysates from cells expressing HA-tagged wild-type Tat (Tat) or mutant Tat (mTat = K41A) were immunoprecipitated with the αCDK7 antibody. One-half of these cell lysates were loaded as input controls (lanes 1,2). Samples were processed and blots probed with αHA antibodies as described above.

Tat associates with recombinant CAK in vitro. Recombinant CAK trimer (50 ng) was incubated with the wild-type (Tat) or mutant (mTat = K41A) Tat proteins (1 μg) bound to streptavidin–agarose beads or with streptavidin–agarose beads alone as the control (C). Nuclear extract (10 μg, lane 1) or CAK (50 ng, lane 2) were loaded as input controls. After washing, the streptavidin beads and controls were subjected to SDS-PAGE, transferred to membranes, and probed with the αcyclin H antibody.

Tat associates with recombinant CDK7 in vitro. CDK7, cyclin H, and MAT 1 (p36) were labeled with ³⁵S by translation in vitro. CDK7 alone (lanes 4–6), in combination with cyclin H (lanes 7–9), or as a mixture with cyclin H and MAT 1 (lanes 10–12), was immunoprecipitated with αCDK7 antibodies bound to protein-A–Sepharose beads. Immunoprecipitates were divided into three fractions. Whereas one-third served as a control for immunoprecipitations (lanes 4,7,10), the remaining two-thirds were incubated with wild-type (T) or mutant (mT = K41A) Tat proteins labeled at their 3′ termini with [γ-³²P]ATP using the catalytic subunit of cAMP-dependent heart muscle kinase. After washing, the beads were subjected to SDS-PAGE. Gels were dried and visualized by autoradiography. One-fifth of the wild-type and mutant Tat proteins used in the binding reactions were loaded as the input controls (lanes 13,14).

Tat increases the ability of CAK to phosphorylate the CTD of Pol II. (A) Wild-type HA-tagged CDK7 (CDK7), or a kinase-deficient mutant of CDK7 (M) were immunoprecipitated from stably expressing HeLa cells. GST–CTD protein (25 ng) was incubated with casein kinase (CK, 25 ng), or CDK7 immunoprecipitates in the presence of wild-type (Tat) or mutant (mTat = K41A) Tat proteins and γ-ATP. Kinase reaction products were subjected to SDS-PAGE, and gels were visualized by autoradiography after drying. Positions of the hypophosphorylated (CTD_a) and hyperphosphorylated (CTD_o) GST–CTD fusions, as well as cyclin H are indicated on the left. (B) GST–CTD was incubated with recombinant CAK (50 ng) in the presence of wild-type (Tat) or mutant (mTat = K41A) Tat proteins in a kinase reaction. Products were processed as described above. (C) Purified Pol II was incubated with CAK in the presence of wildtype (Tat) or mutant (mTat = K41A) Tat proteins in a kinase reaction. Products were processed as described above. (D) CyclinAΔ171/CDK2HA complexes bound to protein-A–Sepharose beads were incubated with CAK (50 ng) in the presence of wild-type (Tat) or mutant (mTat = K41A) Tat proteins. After washing the beads were incubated with histone H1 and kinase reactions allowed to proceed for 1 hr. Products were processed as described above. Position of labeled histone H1 (H1) is indicated.

A CDK2 mutant peptide inhibits the phosphorylation of the CTD by recombinant CAK. (A) Increasing concentrations of a mutant CDK2 peptide (mC2p) were added to kinase reactions containing GST–CTD, and CAK in the presence (+) or absence (−) of Tat. Casein kinase was used as a control for the mobility of the labeled GST–CTD (lane 1). Kinase reactions were processed as described in Fig. 4. Positions of the hypophosphorylated (CTD_a) and hyperphosphorylated (CTD_o) GST–CTD fusions, as well as cyclin H are indicated on the left. (B) The randomized CDK2 peptide (rC2p) was added to kinase reactions as described in A. The labeled GST–CTD fusion protein is presented.

A CDK2 mutant peptide inhibits Tat transactivation. (A) In vitro transcription reactions were done with linearized DNA templates containing the wild-type HIV LTR sequences in the presence (+) or absence (−) of Tat and increasing concentrations of the CDK2 mutant peptide (mC2p). Runoff transcripts (750 nucleotides) were resolved on a 5% polyacrylamide/urea sequencing gel and exposed to x-ray film after drying. (B) Increasing concentrations of the randomized CDK2 peptide (rC2p) were added to in vitro transcription reactions containing the wild-type HIV LTR sequences in the presence (+) or absence (−) of Tat. Runoff transcripts were processed as described in A. (C) Increasing concentrations of the mutant CDK2 peptide (mC2p) were added to in vitro transcription reactions containing linearized DNA templates containing the AdML promoter. Lane 7 was identical to lane 1 except that reactions were done in the presence of α-amanitin (αA = 2 μg/ml). Runoff transcripts (390 nucleotides) were processed as described in A. (D) Increasing concentrations of the mutant CDK2 peptide (mC2p) were added to in vitro transcription reactions containing linearized DNA templates containing the DHFR promoter as described in the experimental procedures. Lane 7 was identical to lane 1 except that reactions were done in the presence of α-amanitin (αA = 2 μg/ml). Runoff transcripts (390 nucleotides) were processed as described in A. The doublet corresponds to alternate transcription start sites. (E) Increasing concentrations of the randomized CDK2 peptide (rC2p) were added to in vitro transcription reactions as described in D.

The mutant CDK2 peptide inhibits Tat transactivation in vivo. (A) Increasing concentrations of the mutant CDK2 peptide (mC2p) were cotransfected into COS cells with a reporter plasmid containing HIV LTR promoter sequences that lacked NF-κB-binding sites (pHIVΔKBCAT) and either functional (+) or nonfunctional (−) Tat. RNase protection assays were done with a probe 220 nucleotides long and hybridized to full-length transcripts of 80 nucleotides (LT) or prematurely terminated transcripts (ST) of 55–59 nucleotides (Okamoto et al. 1996). Protected fragments were resolved on a 11% polyacrylamide/urea sequencing gel and exposed to x-ray film after drying. (B) Increasing concentrations of the randomized CDK2 peptide (rC2p) were cotransfected into COS cells with a reporter plasmid containing the HIV LTR lacking NF-kB-binding sites (pHIVΔKBCAT) and functional Tat. RNase protection assays were done as in A.

Over-expression of CDK7 affects Tat transactivation in vivo. COS cells were cotransfected with HIVSCAT and 4XSp1βglob reporter plasmids, and plasmids encoding HA-tagged functional (+) or nonfunctional (−) Tat, either alone or in combination with wild-type HA-tagged CDK7 (WT) or a kinase-deficient HA-tagged CDK7 (Mut). RNase protection assays were done using a HIV LTR probe that hybridized to full-length LTR–CAT transcripts of 80 nucleotides (LT) or prematurely terminated transcripts (ST) of 55–59 nucleotides (top panel) and a 216 nucleotide β-globin probe that hybridized to transcripts of 179 nucleotides (4XSp1) (Okamoto et al. 1996). (Bottom two panels) Lysates from transfected cells were subjected to SDS-PAGE, transferred to membranes and probed with αHA antibodies to detect Tat–HA and CDK7–HA protein levels.

A model of the mechanism by which Tat increases the processivity of Pol II. As depicted above, transcription from the HIV LTR can be divided into four stages: (1) assembly of the transcription complex onto the DNA template; (2) initiation of transcription; (3) promoter clearance; and (4) elongation of Pol II along the DNA template. In this schematic, the DNA template is depicted by a thick black line, the unphosphorylated form of the core Pol II (IIA) by an open oval with its unphosphorylated CTD as a thin curved line, and the nine subunits TFIIH as striped forms with the CDK7 as a solid circle. Tat is designated as an open circle and the transcription initiation site with an arrow. During assembly of the initiation complex, Tat, TFIIH and other components of the Pol II holoenzyme (omitted for the sake of simplicity) associate with the core Pol II onto the DNA template. Transcription initiates with the hydrolysis of ATP between the β and γ phosphates and the synthesis of the first phosphodiester bond. Nascent RNA (line with a large dot at its 5′ terminus) is synthesized following the promoter clearance. The CTD might be partially phosphorylated at this point and is depicted by a curved line with a partially thickened section. Following synthesis of TAR, Tat binds to the bulge region and is repositioned or modified such that it can increase the ability of TFIIH to phosphorylate the CTD. The highly phosphorylated form of Pol II (IIo), depicted as a shaded oval with its CTD as a thick curved line, is therefore rendered highly processive and can now efficiently transcribe the entire viral genome.