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Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extract extend over the entire transcription unit and approximately 40 base pairs of contiguous 5'-flanking DNA. We show here that the interaction with 5'-flanking DNA is due to a protein that copurifies with transcription factor TFIIIB through several steps of purification and shares characteristic properties that are normally ascribed to TFIIIB: dependence on prior binding of TFIIIC and great stability once the TFIIIC-TFIIIB-DNA complex is formed. SUP4 gene (tRNATyr) DNA that was cut within the 5'-flanking sequence (either 31 or 28 base pairs upstream of the transcriptional start site) was no longer able to stably incorporate TFIIIB into a transcription complex. The TFIIIB-dependent 5'-flanking DNA protein interaction was predominantly not sequence specific. The extension of the transcription complex into this DNA segment does suggest two possible explanations for highly diverse effects of flanking-sequence substitutions on tRNA gene transcription: either (i) proteins that are capable of binding to these upstream DNA segments are also potentially capable of stimulating or interfering with the incorporation of TFIIIB into transcription complexes or (ii) 5'-flanking sequence influences the rate of assembly of TFIIIB into stable transcription complexes.Specific transcription by RNA polymerase III (Pol III) requires the participation of multiple transcription factors. With the exception of the U6 and 7SK RNA genes (7, 14, 17. 41. 50, 53), the DNA-binding sites that anchor these factors are located within transcription units. It is a remarkable property of Pol III that it can transcribe through the bulky transcription complexes that are built up on these internal promoters (also called internal control regions) without dispersing them (69).Specific initiation of transcription at tRNA genes, with which this paper primarily deals, requires factors TFIIIB and -C. Although the yeast analog of TFIIIC (also called T) has been relatively highly purified as a single component (54: see below), HeLa TFIIIC splits into two components upon purification: C2 is the primary DNA-binding determinant, and Cl either modifies the binding properties of C2 or binds to DNA in a C2-dependent manner (12,18,71). Both HeLa C2 factor and yeast TFIIIC (T) are large (12,54,64). The Bonbvx moni tRNA gene transcription factors can also be separated into three fractions, whose relationship to TFIIIB, Cl, and C2 remains to be established (51).The first promoter dissections of Pol III genes identified essential gene-internal elements (10,19,25,55) and implied that flanking DNA sequence was almost without effect on promoter strength. The general situation with regard to the effects of flanking sequence on promoter strength of Pol III genes is, however, diverse, and this fact has only gradually been recognized (3, 4, 20, 29, 52, 62, 63; tions with very substantial effects have been reported. Flanking sequences affecting promoter strength are located within ...

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RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation.