Premature Termination of Tubulin Gene Transcription in Xenopus Oocytes is Due to Promoter-Dependent Disruption of Elongation

Abstract

We have shown previously that the Xenopus α-tubulin gene, XαT14, exhibits premature termination of transcription when injected into oocyte nuclei. The 3′ ends of prematurely terminated transcripts are formed immediately downstream of a stem-loop sequence found in the first 41 bp of the 5′ leader. We show here, using deleted constructs, that premature termination requires the presence only of sequences from -200 to +19 relative to the initiation site. Deletion of the stem-loop does not increase the production of extended transcripts, and premature termination apparently continues at nonspecific sites. This finding indicates that disruption of the elongation phase of transcription rather than abrogation of a specific antitermination mechanism is the cause of premature termination in XαT14. We also found that disruption of elongation on a reporter gene could be induced specifically by competition with XαT14 promoters. To identify which elements of the promoter might interact with elongation determinants to cause this competition, we constructed a series of internal promoter mutants. Most mutations in the -200 to -60 region of the promoter had some effect on initiation frequency but did not cause any significant change in levels of premature termination. However, mutations in the core promoter that removed the TATA box consensus causes major change in initiation and resulted in a marked decrease in the production of prematurely terminated transcripts relative to extended transcripts. We discuss why such promoters can apparently escape the disruption of elongation that leads to premature termination.