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DRB sensitivity-inducing factor (DSIF or Spt4/5) is a conserved transcription elongation factor that both inhibits and stimulates transcription elongation in metazoans. In Drosophila and vertebrates, DSIF together with negative elongation factor (NELF) associates with RNA polymerase II during early elongation and causes RNA polymerase II to pause in the promoter-proximal region of genes. The mechanism of how DSIF establishes pausing is not known. We constructed Spt5 mutant forms of DSIF and tested their capacity to restore promoter-proximal pausing to DSIF-depleted Drosophila nuclear extracts. The C-terminal repeat region of Spt5, which has been implicated in both inhibition and stimulation of elongation, is dispensable for promoter-proximal pausing. A region encompassing KOW4 and KOW5 of Spt5 is essential for pausing, and mutations in KOW5 specifically shift the location of the pause. RNA cross-linking analysis reveals that KOW5 directly contacts the nascent transcript, and deletion of KOW5 disrupts this interaction. Our results suggest that KOW5 is involved in promoter-proximal pausing through contact with the nascent RNA.

The positive transcription elongation factor, P-TEFb, controls the fraction of initiated RNA polymerase II molecules that enter into the productive mode of elongation necessary to generate mRNAs. To better understand the mechanism of this transition into productive elongation we optimized a defined in vitro transcription system and compared results obtained with it to those obtained with a crude system. We found that controlling the function of TFIIF is a key aspect of RNA polymerase II elongation control. Before P-TEFb function, early elongation complexes under the control of negative factors are completely unresponsive to the robust elongation stimulatory activity of TFIIF. P-TEFb-mediated phosphorylation events, targeting the elongation complex containing DSIF and NELF, reverse the negative effect of DSIF and NELF and simultaneously facilitate the action of TFIIF. We also found that productive elongation complexes are completely resistant to negative elongation factors. Our data suggest that an additional factor(s) is involved in establishing the unique resistance activities of the elongation complexes before and after P-TEFb function. Furthermore, we provide evidence for the existence of another positive activity required for efficient function of P-TEFb. A model of the mechanism of P-TEFb-mediated elongation control is proposed in which P-TEFb induces the transition into productive elongation by changing the accessibility of elongation factors to elongation complexes. Our results have uncovered important properties of elongation complexes that allow a more complete understanding of how P-TEFb controls the elongation phases of transcription by RNA polymerase II.

Genetic and proteomic approaches have identified numerous proteins that are potentially involved in regulating transcriptional elongation, but the mechanisms of action of these proteins remain largely unknown. We describe an experimental approach using native gel electrophoresis for studying interactions of elongation factors with isolated Pol II elongation complexes. The gel distinguishes Pol IIA and Pol IIB containing complexes. The interaction of DSIF (Spt4/Spt5) with the elongation complexes can be readily detected, and this association is not dependent on the carboxyl-terminal domain of the largest subunit of Pol II. We also report the surprising observation that a monoclonal antibody that binds the carboxyl-terminal domain of Pol II triggers the dissociation of the elongation complex. The action of the antibody could be mimicking the action of cellular factors involved in transcription termination.

Studying the sensitivity of transcription to the nucleotide analog 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole has led to the discovery of a number of proteins involved in the regulation of transcription elongation by RNA polymerase II. We have developed a highly purified elongation control system composed of three purified proteins added back to isolated RNA polymerase II elongation complexes. Two of the proteins, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), act as negative transcription elongation factors by increasing the time the polymerase spent at pause sites. P-TEFb reverses the negative effect of DSIF and NELF through a mechanism dependent on its kinase activity. TFIIF is a general initiation factor that positively affects elongation by decreasing pausing. We show that TFIIF functionally competes with DSIF and NELF, and this competition is dependent on the relative concentrations of TFIIF and NELF.

Control of transcription elongation requires a complex interplay between the recently discovered positive transcription elongation factor b (P-TEFb) and negative transcription elongation factors, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity inducing factors (DSIF) and the negative elongation factor (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.

5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). We have previously identified and purified a novel transcription elongation factor, termed DSIF (for DRBsensitivity-inducing factor), that makes transcription sensitive to DRB. DSIF is composed of 160- and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4. DSIF may either repress or stimulate transcription in vitro, depending on conditions, but its physiological function remains elusive. Here we characterize the structure and function of DSIF p160. p160 is shown to be a ubiquitous nuclear protein that forms a stable complex with p14 and interacts directly with the pol II largest subunit. Mutation analysis of p160 is used to identify structural features essential for its in vitro activity and to map the domains required for its interaction with p14 and pol II. Finally, a p160 mutant that represses DSIF activity in a dominant-negative manner is identified and used to demonstrate that DSIF represses transcription from various promoters in vivo.