TATA-binding protein and nuclear differentiation in Tetrahymena thermophila.

Abstract

Unambiguous TATA boxes have not been identified in upstream sequences of Tetrahymena thermophila genes analyzed to date. To begin a characterization of the promoter requirements for RNA polymerase II, the gene encoding TATA-binding protein (TBP) was cloned from this species. The derived amino acid sequence for the conserved C-terminal domain of Tetrahymena TBP is one of the most divergent described and includes a unique 20-amino-acid C-terminal extension. Polyclonal antibodies generated against a fragment of Tetrahymena TBP recognize a 36-kDa protein in macronuclear preparations and also cross-react with yeast and human TBPs.Immunocytochemistry was used to examine the nuclear localization of TBP during growth, starvation, and conjugation (the sexual phase of the life cycle). The transcriptionaily active macronuclei stained at all stages of the life cycle. The transcriptionally inert micronuclei did not stain during growth or starvation but surprisingly stained with anti-TBP throughout early stages of conjugation. Anti-TBP staining disappeared from developing micronuclei late in conjugation, corresponding to the onset of transcription in developing macronuclei. Since micronuclei do not enlarge or divide at this time, loss of TBP appears to be an active process. Thus, the transcriptional differences between macro-and micronuclei that arise during conjugation are associated with the loss of a major component of the basal transcription apparatus from developing micronuclei rather than its appearance in developing macronuclei.TATA-binding protein (TBP) is required for transcription by all three nuclear RNA polymerases. TBP, along with an assortment of distinct TBP-associated factors, has been found to make up the RNA polymerase I selectivity factor I (9, 36), the polymerase II general transcription factor IID (14,73,83), and the RNA polymerase III general transcription factor IIIB (38,43,62,72,75). Given the need for TBP to function in these different complexes in such fundamental cellular processes, it is not surprising that TBP is highly conserved throughout eukaryotes. Yeast and human TBP are functionally interchangeable in basal transcription reactions reconstituted with yeast or human components (5,17,39) and have nearly identical TATA element binding specificities (78). The C-terminal 180 residues of TBP from a variety of organisms are typically 80% identical in amino acid sequence, whereas the N-terminal regions are divergent both in length and in amino acid sequence (6, 16, 20, 26, 28-30, 33, 37, 49, 55, 61). The C-terminal domain is necessary and sufficient for TATA element binding and basal transcription in vitro (28,41,55) and for the essential functions of yeast TBP in yeast cells (10,21,57,84). This region forms an independent structural domain within the context of the intact protein (41). It contains an interrupted repeat of 67 amino acids, a short basic repeat, and a region weakly homologous with bacterial sigma factors (10, 80). In addition to its role in the basal transcription apparatus, TBP also ...

Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila.

“…TBP is a general factor required for transcription by all three nuclear RNA polymerases (for review, see White and Jackson 19921 Rigby 1993}. Anti-TBP serum stains only macronuclei during growth and starvation but stains nuclei during conjugation with a pattem similar to that observed for anti-hvll parental macronuclei, meiotic micronuclei, gametic and postzygotic nuclei stain with anti-TBP antibody (Stargell and Gorovsky 1993). Anti-TBP staining disappears from micronuclei and is restricted to developing macronuclei late in development.…”

“…Recent observations show that TATA-binding protein (TBP), a component necessary for transcription by all three nuclear RNA polymerases {for review, see White and Jackson 1992;Rigby 1993}, is acquired by micronuclei just before this period of transcriptional activity (Stargell and Gorovsky 1993}. Anti-TBP antibodies stain micronuclei just before and continue to stain micronuclei after this period of transcriptional activity, whereas micronuclei in starved and growing cells do not stain.…”

Study of an RNA helicase implicates small RNA–noncoding RNA interactions in programmed DNA elimination in Tetrahymena

“…S1), when new Mac development started. This biphasic pattern suggests that ncRNA transcription from IES sequences occurs twice, first at meiotic prophase, the only stage when RNA synthesis and the transcription apparatus are detected in the Mic (Sugai and Hiwatashi 1974;Stargell and Gorovsky 1994;Mochizuki and Gorovsky 2004c), and then when new Macs develop.…”

An unusual fibrillarin gene and protein: structure and functional implications.

“…No TAG codons are found and the translation termination codon is TGA as in all known Tetrahymena genes (Tourancheau et al, 1995). The G+C content of the coding region is 43.4% greater than the 39.5% G+C content of 35 other Tetrahymena coding regions (Stargell and Gorovsky, 1994 …”

“…The 560-bp 3' untranslated region is unusually long for a Tetrahymena gene (Vohra et al, 1992;Stargell and Gorovsky, 1994;Heinonen and Pearlman, 1994). Four possible polyadenylation signals (AATAAA) are present, all of which are further upstream of the poly(A) tail than usually found in Tetrahymena (Vohra et al, 1992;Heinonen and Pearlman, 1994).…”