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Papers In Press, published online ahead of print December 8, 2000
J. Biol. Chem, 10.1074/jbc.M006130200
Submitted on July 12, 2000
Revised on December 8, 2000
Accepted on December 7, 2000
Pharmacology, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854
Corresponding Author: rana{at}umdnj.edu
Control of transcription elongation requires a complex interplay between the recently discovered positive transcription elongation factor b (P-TEFb) and negative transcription elongation factors, DRB-sensitivity-inducing factors (DSIF) and the negative elongation factor complex (NELF). Activation of HIV-1 gene expression is regulated by a nascent RNA structure, termed TAR RNA, in concert with HIV-1 Tat protein and these positive and negative elongation factors. We have used a stepwise RNA Pol II walking approach and Western blotting to determine the dynamics of interactions between HIV-1 Tat, DSIF/NELF, and the transcription complexes actively engaged in elongation. In addition, we developed an in vitro kinase assay to determine the phosphorylation status of proteins during elongation stages. Our results demonstrate that DSIF/NELF associates with RNA pol II complexes during early transcription elongation and travels with elongation complexes as the nascent RNA is synthesized. Our results also show that HIV-1 Tat protein stimulated DSIF and RNA Pol II phosphorylation by P-TEFb during elongation. These findings reveal a molecular mechanism for the negative and positive regulation of transcriptional elongation at the HIV-1 promoter.
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