Nucleic Acids Research, 1994, Vol. 22, No. 16 3433-3439
© 1994
METHODS |
Interactions between yeast TFIIIB components
Service de Biochimie et Génétique Moleculaire CEA-Saclay, F91191 Gif sur Yvette Cedex, France
*To whom correspondence should be addressed
Received March 25, 1994. Revised May 25, 1994. Accepted May 25, 1994.
"Yeast transcription factor TFIIIB is a multicomponent factor comprised of the TATA-binding protein TBP and of associated factors TFIIIB70 and B". Epitope-tagged or histidine-tagged TFIIIB70 could be quantitatively removed from TFIIIB by affinity chromatography. "TBP and B (apparent mass 160–200 kDa) could be easily separated by gel filtration or ion-exchange chromatography. While only weak interactions were detected between TBP and B", direct binding of [35S]-labeled TBP to membrane-bound TFIIIB70 could be demonstrated in absence of DNA. On tRNA genes, there was no basal level of transcription in the complete absence of TBP. "The two characterized TFIIIB components (recombinant rTFIIIB70 and rTBP) and a fraction cochromatographing with B" activity were found to be required for TFIIIC-independent transcription of the TATA-containing U6 RNA gene in vitro. Therefore, beside the TFIIIC-dependent assembly process, each TFIIIB component must have an essential role in DNA binding or RNA polymerase recruitment.
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