The termination of transcription for poly(A) mRNA transcription units in eukaryotes is generally presumed to be a 2 step process in which cleavage/polyadenylation of the RNA (step l) facilitates the actual termination of transcription (step 2) at an appropriate down-stream signal. It is not known whether it is the assembly of a functional cleavage/polyadenylation complex on the RNA, or the reaction that it carries out (cleavage), that confers on the RNA polymerase the predisposition to terminate. This will be tested by inserting self- cleaving RNA sequences into transcription units so as to uncouple cleavage and poly(A) complex assembly. It is also not known whether the two steps are obligatorily coupled. Does cleavage always predispose to termination? This will be tested by functionally characterizing the chicken globin beta-epsilon gene 3 flanking region which appears to be defective in transcription termination. The beta-epsilon region contains what appears to be a transcriptional arrest site which is unable to function as a terminator by releasing the arrested polymerases. Nuclear run-on transcription results indicate that after the polymerases traverse the beta-epsilon gene poly(A) site they stack up behind the arrest site without dissociating efficiently from the template. The poly(A) and arrest sites of the beta-epsilon gene will be tested in combination with poly(A) and pause sites from other genes in order to localize and characterize the defect. The other side of the obligatory coupling question is whether the signals can ever operate independently of each other. The nature of the downstream signal, in particular, remains ambiguous. Transcriptional pausing is likely to be a common denominator of such signals but whether, in special circumstances, some of the signals may be designed as stand-alone terminators remains a significant possibility. A new experimental approach which distinguishes between pausing and termination will be used to determine whether the unusually effective downstream signals of the beta-H terminator can function as stand-alone terminators. It has been postulated that a minimum spacing between the poly(A) site and the down-stream elements is required to facilitate tracking. Preliminary results suggest that the beta-H gene does not have such a requirement. The minimum allowable spacing for the beta-H terminator will be determined to shed light on this issue. A major problem in the study of termination is the lack of suitably convenient techniques. A major goal of this proposal is to refine the design of several novel transfection vectors which have been developed for the study of termination. One is for use in run-on transcription and places two G-free cassettes of different-length under the control of the SV40 early promoter. Insertions are made between the two cassettes and termination is assayed by Tl RNase digesting the run-on transcripts and separating them on a gel (G-free RNA is immune to T1 RNase). A functional terminator will prevent transcription of the second cassette. The other vector is a CAT expression vector that operates on the principle of promoter interference. Terminators are recognized by their ability to rescue CAT expression by terminating transcription from an interfering upstream promoter.
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