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Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR.

Sedore SC, Byers SA, Biglione S, Price JP, Maury WJ, Price DH - Nucleic Acids Res. (2007)

Bottom Line: P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2.Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat.In addition, we found that HEXIM1 binds tightly to the HIV 5' UTR containing TAR and recruits and inhibits P-TEFb activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Iowa, Iowa City, IA, USA.

ABSTRACT
Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb-HEXIM1-7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb-HEXIM1-7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5' UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR.

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HEXIM1 binds TAR and the HEXIM1–TAR complex recruits P-TEFb. The binding of HEXIM1, TAR and P-TEFb was evaluated under stoichiometric conditions by EMSA. The indicated components were pre-incubated and the resulting complexes were resolved by gel electrophoresis on a native gel. The gel was silver-stained to visualize protein shifts and autoradiography used to visualize the 32P-labeled RNA shifts.
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Figure 6: HEXIM1 binds TAR and the HEXIM1–TAR complex recruits P-TEFb. The binding of HEXIM1, TAR and P-TEFb was evaluated under stoichiometric conditions by EMSA. The indicated components were pre-incubated and the resulting complexes were resolved by gel electrophoresis on a native gel. The gel was silver-stained to visualize protein shifts and autoradiography used to visualize the 32P-labeled RNA shifts.

Mentions: Given the previous results demonstrating that HEXIM1 and Tat compete for binding to the same small 7SK oligo, and the similarity of this region with the TAR RNA, we explored the possibility that HEXIM1 might bind to TAR. If this is the case, it might also be able to bind and inhibit P-TEFb much the same way that HEXIM1 and 7SK inhibit P-TEFb, thus repressing P-TEFb, and transcription elongation, at the HIV LTR promoter. To begin testing this hypothesis, EMSAs were used to determine if HEXIM1 could bind to TAR and recruit P-TEFb. 32P-labeled, chemically synthesized TAR RNA was combined with FPLC-purified HEXIM1, and P-TEFb containing Cdk9 and the first 290 amino acids of cyclin T1, as indicated; complexes were resolved on a native gel and both silver-stained and exposed to film to visualize both the RNA and protein shifts (Figure 6). When HEXIM1 and TAR were combined, a new complex migrating more slowly than either HEXIM1 or TAR RNA was observed, suggesting the formation of a HEXIM1-TAR complex (Figure 6, H1–TAR). As P-TEFb was titrated onto this complex, there was a further shift of the complex with more mobility than free P-TEFb, accompanied by a change in the color of the silver-stained gel complex, suggesting the addition of a new component and the formation of a P-TEFb–HEXIM1–TAR complex (Figure 6, H1–P–TAR). More importantly, free P-TEFb, which does not bind to RNA alone, disappeared.Figure 6.


Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR.

Sedore SC, Byers SA, Biglione S, Price JP, Maury WJ, Price DH - Nucleic Acids Res. (2007)

HEXIM1 binds TAR and the HEXIM1–TAR complex recruits P-TEFb. The binding of HEXIM1, TAR and P-TEFb was evaluated under stoichiometric conditions by EMSA. The indicated components were pre-incubated and the resulting complexes were resolved by gel electrophoresis on a native gel. The gel was silver-stained to visualize protein shifts and autoradiography used to visualize the 32P-labeled RNA shifts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935001&req=5

Figure 6: HEXIM1 binds TAR and the HEXIM1–TAR complex recruits P-TEFb. The binding of HEXIM1, TAR and P-TEFb was evaluated under stoichiometric conditions by EMSA. The indicated components were pre-incubated and the resulting complexes were resolved by gel electrophoresis on a native gel. The gel was silver-stained to visualize protein shifts and autoradiography used to visualize the 32P-labeled RNA shifts.
Mentions: Given the previous results demonstrating that HEXIM1 and Tat compete for binding to the same small 7SK oligo, and the similarity of this region with the TAR RNA, we explored the possibility that HEXIM1 might bind to TAR. If this is the case, it might also be able to bind and inhibit P-TEFb much the same way that HEXIM1 and 7SK inhibit P-TEFb, thus repressing P-TEFb, and transcription elongation, at the HIV LTR promoter. To begin testing this hypothesis, EMSAs were used to determine if HEXIM1 could bind to TAR and recruit P-TEFb. 32P-labeled, chemically synthesized TAR RNA was combined with FPLC-purified HEXIM1, and P-TEFb containing Cdk9 and the first 290 amino acids of cyclin T1, as indicated; complexes were resolved on a native gel and both silver-stained and exposed to film to visualize both the RNA and protein shifts (Figure 6). When HEXIM1 and TAR were combined, a new complex migrating more slowly than either HEXIM1 or TAR RNA was observed, suggesting the formation of a HEXIM1-TAR complex (Figure 6, H1–TAR). As P-TEFb was titrated onto this complex, there was a further shift of the complex with more mobility than free P-TEFb, accompanied by a change in the color of the silver-stained gel complex, suggesting the addition of a new component and the formation of a P-TEFb–HEXIM1–TAR complex (Figure 6, H1–P–TAR). More importantly, free P-TEFb, which does not bind to RNA alone, disappeared.Figure 6.

Bottom Line: P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2.Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat.In addition, we found that HEXIM1 binds tightly to the HIV 5' UTR containing TAR and recruits and inhibits P-TEFb activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Iowa, Iowa City, IA, USA.

ABSTRACT
Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb-HEXIM1-7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb-HEXIM1-7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5' UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR.

Show MeSH
Related in: MedlinePlus