Abstract
Human immunodeficiency virus, type 1 (HIV-1) Tat protein activates transcription from the HIV-1 long terminal repeat. Tat interacts with TFIIH and Tat-associated kinase (a transcription elongation factor P-TEFb) and requires the carboxyl-terminal domain of the largest subunit of RNA polymerase II (pol II) for transactivation. We developed a stepwise RNA pol II walking approach and used Western blotting to determine the role of TFIIH and P-TEFb in HIV-1 transcription elongation. Our results demonstrate the new findings that P-TEFb is a component of the preinitiation complex and travels with the elongating RNA pol II, whereas TFIIH is released from the elongation complexes before the trans-activation responsive region RNA is synthesized. Our results suggest that TFIIH and P-TEFb are involved in the clearance of promoter-proximal pausing of RNA pol II on the HIV-1 long terminal repeat at different stages.
Highlights
Transcription elongation in eukaryotic genes is a complex process that involves a number of regulatory factors
Our results demonstrate that P-TEFb is a component of the preinitiation complex and travels with the elongating RNA polymerase II (pol II), whereas TFIIH is released from the elongation complexes before the TAR RNA is synthesized
Immobilized DNA templates with beads upstream of the promoter were used for stepwise walking of RNA pol II, and DNA templates with beads downstream of the promoter were used to stall elongation complexes chased with all four nucleotides
Summary
Transcription elongation in eukaryotic genes is a complex process that involves a number of regulatory factors. 1 The abbreviations used are: pol II, polymerase II; HIV-1, human immunodeficiency virus, type 1; CTD, carboxyl-terminal domain; LTR, long terminal repeat; TAR, trans-activation responsive region; PIC, preinitiation complex; TEC, transcription elongation complex; PAGE, polyacrylamide gel electrophoresis.
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