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.

Marking the start site of RNA polymerase III transcription: the role of constraint, compaction and continuity of the transcribed DNA strand

“…The downstream edge of the TFIIIB footprint on the N−14b probe is obscured by the loss, during native gel electrophoresis, of double‐stranded DNA fragments formed by DNase I‐generated nicks in the labeled non‐transcribed strand within ∼10 bp of the preformed break in the transcribed strand (indicated by hatching in the black box). Enhanced DNase I cleavage around the start site of transcription upon TFIIIB–DNA complex formation has been noted previously (Kassavetis et al ., 1989).…”

“…The purification and quantification of proteins has been described (Kassavetis et al ., 2001). Quantities of pol III are specified as fmol of enzyme active for specific transcription (Kassavetis et al ., 1989); quantities of the other proteins are specified as fmol protein. TBP and Bdp1 were estimated to be nearly 100% active and Brf1 20% active in the formation of heparin‐resistant TFIIIB–DNA complexes.…”

“…Multiple‐round transcription during 30 min was initiated by adding 5 μl of reaction buffer containing 1 mM GTP, ATP and CTP, and 125 μM [α‐ 32 P]UTP (10 c.p.m./fmol). Reactions were stopped, samples were processed for denaturing gel electrophoresis and quantified by phosphor image plate analysis (Kassavetis et al ., 1989). Primer extension analysis of unlabeled transcripts followed Kassavetis et al .…”

Efficient synthesis, termination and release of RNA polymerase III transcripts in Xenopusextracts depleted of La protein

“…To determine if X.laevis La has a transcript release function, we examined the efficiency of pol III transcription in La‐depleted extracts in the presence and absence of heparin to distinguish single from multiple rounds of transcription (Kassavetis et al ., 1989). Several factors were varied in preliminary experiments to optimize this approach.…”

A Position-Dependent Transcription-Activating Domain in TFIIIA

“…One component of TFIIIB is the TATA box-binding protein, which is required for transcription by RNA polymerases I, II, and III (9,29,32,46,49). TFIIIB specifically interacts with an upstream DNA-binding site only after assembly I into the transcription complex (2,27,30). The activation mechanism for DNA binding by TFIIIB is unknown but presumably results from protein-protein interactions.…”