While transcription initiation is the most important single control point in eukaryotic gene expression, the molecular mechanisms involved in promoter recognition and use by RNA polymerase II are not thoroughly understood. We propose here to extend several of our recent studies on initiation and the transition into elongation by RNA polymerase II. We have shown that abortive initiation accompanies productive initiation by the polymerase. The ratio of abortive to productive initiation (that is, the efficiency of promoter clearance) varies over a wide range among various promoters. In some but not all cases the weaker promoters are characterized by less effective clearance. We now propose to address the following questions. Will the use of stimulatory transcription factors increase the efficiency of clearance at otherwise inefficient promoters? Will high levels of some of the core transcription factors suppress abortive initiation? Can we understand the intrinsic differences in abortive initiation among promoters in terms of their sequence and their architecture? In particular, what is the relative role of the TATA box and the initiator element in determining the efficiency of promoter clearance? It had been assumed that once promoter clearance is achieved, the RNA polymerase II transcription complex has a relatively uniform structure throughout elongation. We have recently shown that this is not the case, either structurally or in terms of stability, for highly purified complexes paused at two different locations during elongation. We will assemble and characterize other paused elongation complexes, including complexes paused at natural pause sites, in order to determine whether structural features can be correlated with a high probability of pausing or low complex stability. We have also shown that a sequence which should be recognized as a pause site during elongation does not cause significant pausing when placed very close to the point of transcription initiation. We will construct a series of promoters in which potential pause sites are placed at increasing distances from the initiation site in order to map the minimum extent of elongation required for pause sites to become effective. Finally, we will attempt to map regions within the RNA polymerase itself which are important for initiation, clearance and effective elongation. These collaborative studies will involve characterizing the behavior, particularly at initiation and clearance, of existing temperature-sensitive yeast RNA polymerases as well as the generation and characterization of new mutations within yeast RNA polymerase II.
Showing the most recent 10 out of 37 publications
