Properties of RNA Polymerase II Elongation Complexes Before and After the P-TEFb-mediated Transition into Productive Elongation

Abstract

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.