Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.
Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.
Ducrot
Cécile
C
Service de Biochimie et de Génétique Moléculaire, Bâtiment 144, CEA/Saclay, F-91191 Gif-sur-Yvette Cedex, France.
Lefebvre
Olivier
O
Landrieux
Emilie
E
Guirouilh-Barbat
Josée
J
Sentenac
André
A
Acker
Joel
J
D016367:Baculoviridae / Q000235:genetics; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins; D015966:Gene Expression Regulation, Fungal; D046912:Multiprotein Complexes; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012320:RNA Polymerase III / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D035361:Transcription Factor TFIIIB / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019474:Transcription Factors, TFIII / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D000098832:Transcription Factor TFIIIC
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins; D035361:Transcription Factor TFIIIB; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D012320:RNA Polymerase III