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 ...

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