Expression, proteolytic analysis, reconstitution, and crystallization of the tau60/tau91 subcomplex of yeast TFIIIC

Protein Expr Purif. 2006 Feb;45(2):255-61. doi: 10.1016/j.pep.2005.06.013. Epub 2005 Jul 26.

Abstract

The transcription factor IIIC (TFIIIC) is a multisubunit DNA-binding factor required for promoter recognition and TFIIIB assembly on tRNA genes transcribed by RNA polymerase III. Yeast TFIIIC consists of six subunits, organized in the two globular subcomplexes tauA and tauB, which recognize two internal tDNA promoter elements, the A and the B block, respectively. As a first step toward a detailed structural analysis of TFIIIC, we report here the expression, proteolytic analysis, reconstitution, and crystallization of the complex between yeast TFIIIC subunits tau91 and tau60. Proteolysis provided an insight into the domain structure of tau60 and tau91. Both the proteins form a stable complex that does not require an N-terminal, protease-sensitive extension of tau91. Crystals diffracting beyond 3.2 A were obtained from a complex formed by full-length tau60 and the N-terminally truncated form of tau91 lacking this extension.

MeSH terms

  • Crystallization
  • Multiprotein Complexes
  • Promoter Regions, Genetic
  • Protein Subunits* / chemistry
  • Protein Subunits* / genetics
  • Protein Subunits* / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae Proteins* / chemistry
  • Saccharomyces cerevisiae Proteins* / genetics
  • Saccharomyces cerevisiae Proteins* / metabolism
  • Transcription Factors, TFIII* / chemistry
  • Transcription Factors, TFIII* / genetics
  • Transcription Factors, TFIII* / metabolism

Substances

  • Multiprotein Complexes
  • Protein Subunits
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors, TFIII
  • transcription factor TFIIIC