RNA POL II POLY-A SITE AND 3' TERMINATION

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The ability to regulate the mRNA population is a key element of normal cell function, required for cell cycle regulation, cell differentiation and cell determination. Minor mRNA changes can have a major impact on cell function, possibly best represented by the effects of altered oncogene expression or hormone induction. Clearly, promoter activation effected by both initiation and repression functions is a dominant player in mRNA metabolism, but it is not the only level at which mRNA populations can be controlled. A growing number of transcription units are also under the influence of "postinitiation" control functions which include premature termination (c-myc, c-myb, Ad-mlp), alternative splicing and alternative polyadenylation (Ad-mlp, calcitonin/CGRP, muscle proteins) and transcription termination (Ig mu-delta, Ad-mlp. In addition to these nuclear events, in the cytoplasm cell specific control of mRNA 1/2 life is also becoming more and more apparent. The "postinitiation" regulation of a cells mRNA population is the general area of research my group is addressing. This proposal deals specifically with experiments designed to understand the mechanisms which operate to regulate two of these events, poly(A) choice in complex transcription units and termination of RNA polymerase II transcription units. One important aspect of postinitiation functions which is presently open to debate is whether the regulation of these events is mediated directly through the RNA polymerase II elongation complex or are these events (particularly splicing and polyadenylation) controlled at a point which is uncoupled from the transcription complex. This issue is also important to the control of transcription termination, since we have demonstrated the DNA sequence AATAAA (polyadenylation signal sequence) is a required cis element of 3' termination. This proposal is directly answering this synthesis (using reconstructed adenovirus vectors) and in vitro dissection of the biochemical process involved in controlling the 3' postinitiation events. Basic information which will be generated by these studies will include comparison of in vivo and in vitro poly (A) site utilization for several polyadenylation signal elements, relate the efficiency of poly (A) site utilization to transcription termination, identify the 3' consensus sequence required to inducer transcription complex displacement and finally, demonstrate how for the adenovirus major late transcription unit, the generation of varied preinitiation complexes at the mlp can influence elongation and processing events at the 3' end.