FIG. 7.
The C-terminal region of CycT1 is required for efficient HIV-1 transcriptional elongation. (A) P-TEFb complexes containing either HA-tagged CycT1Δ1 or CycT1FL were normalized for their Cdk9 and CycT1 levels by Western blotting with anti-Cdk9 and anti-HA antibodies. (B) Equal amounts of the two P-TEFb complexes were added to transcription reaction mixtures containing P-TEFb-depleted HeLa nuclear extracts (NE) as well as the DNA templates pHIV+TAR-G400 and pHIVΔTAR-G100 in the presence (+) or absence (−) of Tat protein. The amount of P-TEFb analyzed in lanes 3 to 6 was three times (3×) higher than that in lanes 7 to 10. +TAR-G400 and ΔTAR-G100 are RNase T1-resistant RNA fragments transcribed from the two G-less DNA cassettes (400 and 100 bp) inserted, respectively, into pHIV+TAR-G400 and pHIVΔTAR-G100 at a position ≈1 kb downstream of the HIV-1 promoter region.