Characterization of the nucleotide requirement for elimination of the rate-limiting step in 5 S RNA gene transcription

We previously demonstrated that the rate-limiting step in 5 S RNA transcription involves the ordered assembly of a preinitiation complex on the 5 S gene that requires magnesium and ATP for its formation and contains stoichiometric amounts of transcription factors IIIA, IIIB, and IIIC and RNA polymerase III. In this paper, we have characterized the nucleotide requirement in a number of ways by taking advantage of our ability to monitor each step in this ordered assembly of transcription components and by direct isolation of transcription complexes. By kinetic analyses, we demonstrate that the lag in transcription can be eliminated by the presence of any nucleoside triphosphate and does not require an energy-yielding event and that the nucleotide is required stoichiometrically. By competition analysis of factor IIIB interaction, we demonstrate that a complete stable complex (which includes factors IIIA, IIIB, and IIIC) can form on the 5 S gene in the absence of nucleotide. Finally, by direct isolation of functional transcription complexes via gel filtration, we demonstrate that RNA polymerase III can also associate with the DNA template in the absence of nucleotide. Thus, the nucleotide requirement for elimination of the rate-limiting step is uncoupled from the actual binding of the transcription components. Further analyses indicate that, consistent with its stoichiometry requirement, the nucleotide can be found associated with the isolated complexes. These results raise the interesting possibility that the nucleotide interaction may be activating the transcription complex via induction of a conformational change in one of the factors or by a general rearrangement of the transcriptional complex on the 5 S gene.