Expression, proteolytic analysis, reconstitution, and crystallization of the tau60/tau91 subcomplex of yeast TFIIIC.
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.
Mylona
Anastasia
A
European Molecular Biology Laboratory, Grenoble Outstation, B.P. 181, 38042 Grenoble Cedex 9, France.
Acker
Joël
J
Fernández-Tornero
Carlos
C
Sentenac
André
A
Müller
Christoph W
CW
D003460:Crystallization; D046912:Multiprotein Complexes; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019474:Transcription Factors, TFIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000098832:Transcription Factor TFIIIC
D046912:Multiprotein Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC