TATA-Binding Protein–TATA Interaction Is a Key Determinant of Differential Transcription of Silkworm Constitutive and Silk Gland-Specific tRNA^Ala Genes

Abstract

We have investigated the contribution of specific TATA-binding protein (TBP)-TATA interactions to the promoter activity of a constitutively expressed silkworm tRNA C Ala gene and have also asked whether the lack of similar interactions accounts for the low promoter activity of a silk gland-specific tRNA SG Ala gene. We compared TBP binding, TFIIIB-promoter complex stability (measured by heparin resistance), and in vitro transcriptional activity in a series of mutant tRNA C Ala promoters and found that specific TBP-TATA contacts are important for TFIIIB-promoter interaction and for transcriptional activity. Although the wild-type tRNA C Ala promoter contains two functional TBP binding sequences that overlap, the tRNA SG Ala promoter lacks any TBP binding site in the corresponding region. This feature appears to account for the inefficiency of the tRNA SG Ala promoter since provision of either of the wild-type TATA sequences derived from the tRNA C Ala promoter confers robust transcriptional activity. Transcriptional impairment of the wild-type tRNA SG Ala gene is not due to reduced incorporation of TBP into transcription complexes since both the tRNA C Ala and tRNA SG Ala promoters form transcription complexes that contain the same amount of TBP. Thus, the deleterious consequences of the lack of appropriate TBP-TATA contacts in the tRNA SG Ala promoter must come from failure to incorporate some other essential transcription factor(s) or to stabilize the complete complex in an active conformation.The silkworm Bombyx mori provides a clear example of regulated tRNA gene expression. The demand for fibroin, the principal protein of silk, requires highly efficient transcription and translation of the fibroin gene in cells of the silk gland. Translational efficiency in these cells is maximized by the quantitative adaptation of the tRNA population to the composition of fibroin: 44% glycine, 29% alanine, and 12% serine (5,29,30,42). In the case of tRNA Ala , enrichment is achieved both by increasing the level of the constitutive type of tRNA Ala (tRNA C Ala ) and by synthesizing an additional, silk gland-specific, type (tRNA SG Ala ) (31,44). In vitro studies of representative tRNA C Ala and tRNA SG Ala genes have revealed transcription properties consistent with the patterns of tRNA C Ala and tRNA SG Ala accumulation in vivo. That is, in a variety of extracts from non-silk gland cells, the tRNA C Ala gene directs transcription much more efficiently than does the tRNA SG Ala gene, but in concentrated extracts from silk gland, the two genes are equally efficient (60). To understand how these two genes are differentially regulated, we have investigated the basis of the transcriptional impairment of the tRNA SG Ala gene that is observed under typical in vitro conditions. Transcription of silkworm tRNA Ala genes is driven by both internal and external promoter elements (26,36,56). The critical difference between the tRNA C Ala and tRNA SG Ala genes is in the interaction of their 5Ј flanking promoter elements with the transcri...

A Composite Upstream Sequence Motif Potentiates tRNA Gene Transcription in Yeast

“…the 2 35 TTTTTC element in C. elegans), even though TATA-like sequences might be present and play important roles in special cases. 5,6 Also, completely new blocks of sequence conservation, located near position 2 20, appear in insect and mammals, as exemplified by the nearly identical A-rich motif found at position 2 22 upstream of mouse and human tDNAs. Because of its striking evolutionary conservation, such a motif is likely to play an important role in mammalian tRNA gene transcription.…”

“…The differential in vitro activity of silkgland-specific and constitutive tRNA Ala genes in the silkworm, Bombyx mori, has long been known to be due to sequence elements upstream of the TSS, 4 and it has later been shown to rely on the interaction of the TBP component of TFIIIB with a TATA-like element upstream of the constitutive gene. 5,6 Recent analyses have further revealed the presence of TATA-like elements upstream of many Saccharomyces cerevisiae tDNAs, and of the majority of Arabidopsis thaliana and Schizosaccharomyces pombe tDNAs. 7,8 The presence of upstream TATA elements is thus not restricted to particular class III genes, such as SNR6 (coding for the U6 snRNA), that have long been known to support TFIIIC-independent TFIIIB recruitment in vitro.…”

Recruitment of RNA polymerase III to its target promoters

“…All three of these promoter elements are required for efficient transcription in vivo (Brow and Guthrie 1990;Eschenlauer et al 1993). Other examples include some A-and B-box-containing tRNA genes in plants (Yukawa et al 2000), yeast (Dieci et al 2000), and silkworm (Ouyang et al 2000), in which TATA boxes present in the 5Ј-flanking region greatly contribute to transcription efficiency. More recently, an analysis in Schizosaccharomyces pombe has revealed that in this organism nearly all tRNA and 5S genes contain a TATA box upstream of the transcription start site that is required for transcription (Hamada et al 2001).…”

Molecular organization of the 5S rDNA gene type II in elasmobranchs

“…There are hybrid promoters like the U6 snRNA gene in S. cerevisiae that contain both internal promoters and a TATA box 61,62 which contributes to better transcription. 63,64,65 Microsatellites -The different microsatellites arrangements between genera at the NTS region are the main contributors to the variation in the NTS length. It has been shown that there is a correlation between the length of the NTS region and the number of motif repetitions within each region of microsatellites in Rajiformes.…”