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 a-tubulin gene, XcxT14, 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 XaT14. We also found that disruption of elongation on a reporter gene could be induced specifically by competition with XaT14 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 caused maijor changes 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.

Ovary-specific expression of a gene encoding a divergent α-tubulin isotype in Xenopus

“…The promoter of the a-tubulin gene XaT14 is extremely active in the oocyte injection assay [18] and possesses consensus binding sites for many different transcriptional activators, including a heat shock element and a reverse CCAAT box (though not a Y-box). Linker scanner mutations that replace any one or two of the binding sites result in only minor reductions in promoter activity [14], consistent with the complex promoter likely for a gene that is specifically expressed in a large number of different cell types [ 181. Experimental identification of the promoter elements normally required for ovarian expression of XaT14 may therefore be difficult because of the large variety of transcription factors stored in oocytes for later use in developing embryos.…”

“…Morgan, unpublished observations). Previous work with XaT14 [14,18,191 has suggested that the extreme efficiency of its complex promoter in injected oocytes is a fortuitous consequence of its containing many potential binding sites for transcriptional activators that presumably enable promoter activity in diverse cell types. The store of varied transcription factors stockpiled in the oocyte for embryonic development could thus impart unusually high activity to XaT14 promoters upon injection.…”

“…Transcriptional control of a number of Xenopus genes that are expressed during oogenesis as well as in other tissues has been studied by microinjectin cloned include hsp 70 [2], TFIIIA [15, 261 and an a-tubulin gene, XaT14 [14,181. Analysis of the hsp 70 promoter [2] has identified an element required for oocyte-specific transcription that is a type of reverse CCAAT motif termed the Y-box [6].…”

Control of transcription arrest in intron 1 of the murine adenosine deaminase gene.

“…Along similar lines, Meulia et al have reported that mutations altering the c-myc P2 TATA box had two effects: a decrease in overall transcription and a decrease in transcription termination (41). In the Xenopus cx-tubulin gene, mutations which disrupted the TATA box also led to a decrease in transcription and a decrease in the number of prematurely terminated transcripts (17). One hypothesis put forward by both groups centers on the fact that overall levels of transcription in these mutants were substantially reduced.…”

Evolution, Organization, and Expression of α-Tubulin Genes in the Antarctic Fish Notothenia coriiceps

“…Less is known regarding regulation of tubulin gene expression in vertebrates. TATA boxes are generally present in vertebrate ␣-and ␤-tubulin promoters (29,30), and high level expression of a Xenopus ␣-tubulin gene, X␣T14, in oocytes is regulated by three CCAAT boxes, a "heat-shock-like" element (all located 60 -200 bp upstream of the transcription start site), and their corresponding transcription factors (31). Cotranslational regulation of tubulin mRNA stability also contributes to control of cellular tubulin levels (32).…”