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Elongin is a transcription elongation factor that was first identified in mammalian systems and is composed of the three subunits, elongin A, B, and C. Sequence homologues of elongin A and elongin C, but not elongin B, were identified in the yeast genome. Neither yeast elongin A nor C sequence homologues was required for cell viability. The two gene products could be purified from yeast as a complex. A recombinant form of the complex, which could only be produced in bacteria if the gene products were co-expressed, was purified over several chromatographic steps. The complex did not stimulate transcription elongation by yeast RNA polymerase II. Using limited proteolysis, the N-terminal 144 residues of yeast elongin A were shown to be sufficient for interaction with yeast elongin C. The purified complex of yeast elongin C/elongin A(1-143) was analyzed using circular dichroism and nuclear magnetic spectroscopy. These studies revealed that yeast elongin A is unfolded but undergoes a dramatic modification of its structure in the presence of elongin C, and that elongin C forms a stable dimer in the absence of elongin A.

TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter-proximal sites. TFIIH possesses three known ATP-dependent activities: a 3' --> 5' DNA helicase catalyzed by its XPB subunit, a 5' --> 3' DNA helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) kinase activity catalyzed by its CDK7 subunit. In this report, we exploit TFIIH mutants to investigate the contributions of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape by RNA polymerase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.

Transcription factor (TF) IIF is a multifunctional RNA polymerase II transcription factor that has well established roles in both transcription initiation, where it functions as a component of the preinitiation complex and is required for formation of the open complex and synthesis of the first phosphodiester bond of nascent transcripts, and in transcription elongation, where it is capable of interacting directly with the ternary elongation complex and stimulating the rate of transcription. In this report, we present evidence that TFIIF is also required for efficient promoter escape by RNA polymerase II. Our findings argue that TFIIF performs dual roles in this process. We observe (i) that TFIIF suppresses the frequency of abortive transcription by very early RNA polymerase II elongation intermediates by increasing their processivity and (ii) that TFIIF cooperates with TFIIH to prevent premature arrest of early elongation intermediates. In addition, our findings argue that two TFIIF functional domains mediate TFIIF action in promoter escape. First, we observe that a TFIIF mutant selectively lacking elongation activity supports TFIIH action in promoter escape, but is defective in suppressing the frequency of abortive transcription by very early RNA polymerase II elongation intermediates. Second, a TFIIF mutant selectively lacking initiation activity is more active than wild type TFIIF in increasing the processivity of very early elongation intermediates, but is defective in supporting TFIIH action in promoter escape. Taken together, our findings bring to light a function for TFIIF in promoter escape and support a role for TFIIF elongation activity in this process.

We previously identified and purified Elongin by its ability to stimulate the rate of elongation by RNA polymerase II in vitro (Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1993) J. Biol. Chem. 268, 25587-25593). In this report, we present evidence that stimulation of elongation by Elongin requires that the early RNA polymerase II elongation complex undergoes conversion to an Elongin-activable form. We observe (i) that Elongin does not detectably stimulate the rate of promoter-specific transcription initiation by the fully assembled preinitiation complex and (ii) that early RNA polymerase II elongation intermediates first become susceptible to stimulation by Elongin after synthesizing 8-9-nucleotide-long transcripts. Furthermore, we show that the relative inability of Elongin to stimulate elongation by early elongation intermediates correlates not with the lengths of their associated transcripts but, instead, with the presence of transcription factor IIF (TFIIF) in transcription reactions. By exploiting adenovirus 2 major late promoter derivatives that contain premelted transcriptional start sites and do not require TFIIF, TFIIE, or TFIIH for transcription initiation, we observe (i) that Elongin is capable of strongly stimulating the rate of synthesis of trinucleotide transcripts by a subcomplex of RNA polymerase II, TBP, and TFIIB and (ii) that the ability of Elongin to stimulate synthesis of these short transcripts is substantially reduced by addition of TFIIF to transcription reactions. Here we present these findings, which are consistent with the model that maximal stimulation of elongation by Elongin requires that early elongation intermediates undergo a structural transition that includes loss of TFIIF.