Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes.
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.
Kassavetis
G A
GA
Department of Biology, University of California, San Diego, La Jolla 92093.
Riggs
D L
DL
Negri
R
R
Nguyen
L H
LH
Geiduschek
E P
EP
D001483:Base Sequence; D002846:Chromatography, Affinity; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D012320:RNA Polymerase III / Q000378:metabolism*; D012331:RNA, Fungal / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D014158:Transcription, Genetic; D014227:Triazines; D000098832:Transcription Factor TFIIIC
D004271:DNA, Fungal; D005656:Fungal Proteins; D012331:RNA, Fungal; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D014227:Triazines; D000098832:Transcription Factor TFIIIC; C012842:Cibacron Blue F 3GA; D012343:RNA, Transfer; D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III; D003850:Deoxyribonuclease I