Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

Abstract

Constitutive and silk gland-specific tRNAAI" genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNAVa, directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA,. We think that the inefficiency of the tRNAsAG gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNASAGa promoter interacts poorly, relative to the tRNAA'a promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNASAsG gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNAA3la and tRNASAsG transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNASAG gene, we measured competition by templates for defined fractions of this machinery.We find that the tRNAsG gene is greatly impaired, in comparison with the tRNAcA'a gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNAAa and tRNAtIa genes. These results suggest that differential interaction with TFIIIB or

Silkworm TFIIIB Binds both Constitutive and Silk Gland-Specific tRNAAla Promoters but Protects Only the Constitutive Promoter from DNase I Cleavage

“…Under these conditions, Ͼ90% of the 8-nucleotide nascent transcripts are correctly elongated and terminated (52,58). Thus, the molar amount of transcript is a direct measure of the molar amount of active transcription complex.…”

“…Fractions of the B. mori class III transcription machinery. TFIIIC/D was isolated as described elsewhere (52). TFIIIB was isolated from the DEII fraction by a modification of a method described previously (34) that yields highly concentrated TFIIIB activity, free of detectable RNA polymerase III.…”

“…We compared the abilities of tRNA C Ala and tRNA SG Ala genes to compete with a reference gene for the limiting component in each case (52). These experiments showed that, compared with the tRNA C Ala gene, the tRNA SG Ala gene was somewhat impaired in its ability to compete for all components but that it was a particularly weak competitor for TFIIIB or RNA polymerase III.…”

“…Moreover, since DNA is not assembled into chromatin in any of these extracts (68), it is unlikely that differential in vitro transcription of tRNA C Ala and tRNA SG Ala genes is mediated through chromatin structure. Single-round transcription assays showed that the difference between tRNA C Ala and tRNA SG Ala transcription rates is quantitatively accounted for by the numbers of active transcription complexes formed on each kind of template (52 complexes. Once formed, complexes on both genes direct multiple rounds of transcription at the same rate.…”

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

“…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 transcription factor complex, TFIIIB (47). Although the tRNA SG Ala gene can direct the addition of TFIIIB to a TFIIIC/ D-promoter complex, as judged by band shift, upstream extension of the TFIIIC/D footprint is not observed when TFIIIB binds the tRNA SG Ala promoter as it is when TFIIIB binds the tRNA C Ala promoter (59).…”