Engineering dimer-stabilizing mutations in the TATA-binding protein.
The TATA-binding protein (TBP) plays a central role in assembling eukaryotic transcription complexes and is subjected to extensive regulation including auto-inhibition of its DNA binding activity through dimerization. Previously, we have shown that mutations that disrupt TBP dimers in vitro have three detectable phenotypes in vivo, including decreased steady-state levels of the mutants, transcriptional derepression, and toxicity toward cell growth. In an effort to more precisely define the multimeric structure of TBP in vivo, the crystallographic dimer structure was used to design mutations that might enhance dimer stability. These mutations were found to enhance dimer stability in vitro and significantly suppress in vivo phenotypes arising from a dimer-destabilizing mutation. Although it is conceivable that phenotypes associated with dimer-destabilizing mutants could arise through defective interactions with other cellular factors, intragenic suppression of these phenotypes by mutations designed to stabilize dimers provides compelling evidence for a crystallographic dimer configuration in vivo.
Kou
Haiping
H
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 452 N. Frear Laboratory, University Park, PA 16802, USA.
Pugh
B Franklin
BF
D019943:Amino Acid Substitution; D001120:Arginine; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D035181:TATA-Box Binding Protein / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D035181:TATA-Box Binding Protein; D001120:Arginine