Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors.

Abstract

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNAAIaC gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNAAIaC promoter is in contact with factors. The protected region extends from -1 to at least + 136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequencespecific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNAAlaC promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.The promoters of tRNA genes have generally been thought to consist of two small segments internal to the coding region. These promoter elements are each about 10 bp long and are called the A and B boxes (reviewed in reference 18). This picture was originally derived from studies of the transcriptional properties of mutant tRNA genes that had undergone partial deletion (8,16,34). It has been supported by the effects of some point mutations within the A and B boxes (reviewed in reference 18). The idea of a two-element tRNA promoter gained additional appeal from early reports that two distinct transcription factor fractions were required, along with polymerase III, for tRNA transcription. A straightforward synthesis of these data led to a model for tRNA transcription in which two transcription factors, called TFIIIB and TFIIIC, activate tRNA genes by binding to the A and B boxes, respectively (11,28,35; see also reference 25).In contrast to this view, work from our laboratory shows that the full tRNA promoter extends far beyond the A and B boxes and that the machinery that acts upon it includes more than two transcription factors. In particular, DNA sequences completely outside the coding region of a Bombyx silkworm tRNAA'IaC gene are required for full transcriptional activity in vitro (40). The promoter for this gene occupies a region of -160 contiguous bp, including the entire coding region plus at least 13 bp upstream of the transcription start site and at least 48 bp downstream of the transcription termination site. Moreover, we have shown that in the case of the silkworm, the transcription apparatus consists of at least four components, each of which is absolutely required (29). These components are polymerase III and transcription factors TFIIIB, TFIIIC, and TFIIID.Several pieces of ...

tRNA genes as transcriptional repressor elements.

“…TFIIIB is composed of several subunits, including the TATA-binding protein (3,40,50,53,66,67,92,103,104). If this factor, or any other component of the pol III complex (7,9,17,19,21,48,51,63,91,105), bound tightly to the activation domains of factors bound at the UAS elements, interactions with the pol II basal promoter complex could be competed for (Fig. 7, model A).…”

A Carboxy-Terminal Basic Region Controls RNA Polymerase III Transcription Factor Activity of Human La Protein

“…Facilitated recycling by yeast pol III onto the same template is dependent on the presence of downstream sequences that include the pol III terminator (14). Likewise, downstream sequences are required for efficient assembly and utilization of yeast, insect, and mammalian transcription complexes (2,43,47). The requirement for downstream sequences including a functional pol III terminator in facilitated recycling suggests that proper termination plays a role in restoring reinitiation competence to the exiting polymerase and/or the transcription complex, although other explanations are possible and this has not been rigorously examined.…”

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

“…When gel-resolved complexes were footprinted, the binding reaction mixture was treated with DNase as described for direct footprinting but digestion was stopped by adding EDTA to a final concentration of 5 mM and immediately fractionating the sample on the 3.5%-10% polyacrylamide step gel described in the paragraph above. Protein-DNA complexes were detected autoradiographically and the DNA fragments were isolated and run on sequencing gels, as described previously (67).…”

“…Template DNA was typically supercoiled, but linear and supercoiled templates are transcribed with equal efficiency by both crude and fractionated silkworm transcription machineries. Single-round transcription assays were performed under the same conditions, with the addition of heparin, as described elsewhere (67), except that both tRNA C Ala -and tRNA SG Ala -promoted reactions were initially stalled by omission of the same nucleotide, CTP, and both transcripts were labelled with [␣-32 P]UTP. This protocol eliminates potential differences in stalled complex formation due to differences in the location of the complexes and was made possible by using an altered chimeric tRNA SG Ala -tRNA C Ala gene whose primary transcript is identical to that of a tRNA C Ala gene.…”