Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes.

Abstract

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 ...

Interactions between yeast TFIIIB components

“…The B' fraction is capable of assembling into the TFIlC * 3tDNA complex, and the incorporation of B" to the B' * 3TFlIC * 3tDNA complex confers the ability to recruit the RNA polymerase for transcription initiation (20). Remarkably, after being assembled, the TF -B-3DNA complex becomes high salt and heparin-resistant under conditions that dissociate TFLIC (3). The TBP is retained in the heparin-resistant complex (5,6,16).…”

“…When bound, TFIHC recruits factor TFIB, which by itself does not bind detectably DNA. Once assembled upstream of the transcriptional start site, TFIIB is sufficient to position RNA polymerase III for transcription initiation (2,3,4). The TATA-binding protein (TBP) is part of TFIB and is involved in this multistep assembly process (5,6).…”

RNA polymerase III interferes with Ty3 integration

“…Ty3 was under the transcriptional control of the GAL1-10 UAS to confer galactose inducibility upon Ty3. The plasmid pTZl carried the SUP4 tRNA 1 ''"' with a G62C mutation that increased promoter efficiency [32].…”

“…A yeast extract which supported transcription by pol III was generated according to the method of Klekamp and Weil [33], with modifications introduced by Kassavetis et al [32]. Yeast strain yTM442 was grown to OD 60 o of 3-5 in YPD (2% yeast extract, 1% peptone, 2% dextrose).…”

Mutational analysis of the transcription start site of the yeast tRNALeu3gene

“…It is both necessary and sufficient to direct Pol III to the transcriptional start site of tRNA genes (10). TFIIIB appears not to recognize specific sequences, but to be directed to its 40 bp long DNA binding site, upstream of the transcriptional start site, by intragenically bound TFIIIC (11 We analyzed a large collection of S.cerevisiae tRNA genes to derive a conserved sequence element (CSE) in the neighborhood of the transcriptional start site (Fig. IA), as others have done previously (16)(17).…”