Transcription initiated by RNA polymerase II and purified transcription factors from liver. Transcription factors alpha, beta gamma, and delta promote formation of intermediates in assembly of the functional preinitiation complex

Abstract

Accurate initiation at promoters by RNA polymerase II in a highly purified transcription system from rat liver depends on five accessory factors, which comprise two functional classes: (i) "promoter recognition" factors, designated tau and epsilon, which interact with template DNA to form an initial complex that serves as a recognition site for binding by RNA polymerase II and (ii) "RNA chain initiation" factors, designated alpha, beta gamma, and delta, which do not participate in initial complex formation, but which are essential for initiation (Conaway, J. W., Reines, D., and Conaway, R. C. (1990) J. Biol. Chem. 265, 7552-7558). Here we investigate the roles of alpha, beta gamma, and delta in accurate initiation. Kinetic evidence indicates that all three factors act in a stage prior to RNA synthesis to facilitate formation of a functional preinitiation complex. Moreover, results of "template challenge" experiments argue that all three factors become stably associated with the preinitiation complex during this stage. Neither alpha, beta gamma, nor delta functions catalytically in this process; instead, each factor appears to interact directly and stoichiometrically with intermediates in assembly of the preinitiation complex. Order of addition experiments reveal that transcription factors alpha and beta gamma assemble into the preinitiation complex by an "ordered" mechanism. We discuss two recently proposed models for assembly of the functional preinitiation complex and argue that our findings provide a plausible means of reconciling them.