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.

[1]  J. W. Rooney,et al.  SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. , 1992, Genes & development.

[2]  B. Pugh,et al.  Dimerization of the TATA Binding Protein (*) , 1995, The Journal of Biological Chemistry.

[3]  M. Muramatsu,et al.  Multimerization of the mouse TATA-binding protein (TBP) driven by its C-terminal conserved domain. , 1994, Nucleic acids research.

[4]  Steven Hahn,et al.  Crystal structure of a yeast TBP/TATA-box complex , 1993, Nature.

[5]  L. Kay,et al.  Solution Structure of a TBP–TAFII230 Complex Protein Mimicry of the Minor Groove Surface of the TATA Box Unwound by TBP , 1998, Cell.

[6]  B. Pugh,et al.  TFIIA regulates TBP and TFIID dimers. , 1999, Molecular cell.

[7]  Madhusmita Mitra,et al.  Structural and Functional Analysis of Mutations along the Crystallographic Dimer Interface of the Yeast TATA Binding Protein , 2003, Molecular and Cellular Biology.

[8]  B. Pugh,et al.  Control of gene expression through regulation of the TATA-binding protein. , 2000, Gene.

[9]  K. Struhl,et al.  Multiple functions of the nonconserved N-terminal domain of yeast TATA-binding protein. , 2001, Genetics.

[10]  B. Pugh,et al.  A genome-wide housekeeping role for TFIID and a highly regulated stress-related role for SAGA in Saccharomyces cerevisiae. , 2004, Molecular cell.

[11]  P. Sharp,et al.  Crystal structure of yeast TATA-binding protein and model for interaction with DNA. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[12]  B. Pugh,et al.  Interplay of TBP inhibitors in global transcriptional control. , 2002, Molecular cell.

[13]  K. Struhl.  Fundamentally Different Logic of Gene Regulation in Eukaryotes and Prokaryotes , 1999, Cell.

[14]  M. Horikoshi,et al.  The conserved carboxy-terminal domain of Saccharomyces cerevisiae TFIID is sufficient to support normal cell growth , 1991, Molecular and cellular biology.

[15]  R Ohba,et al.  Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. , 1997, Genes & development.

[16]  B. Pugh,et al.  A role for TBP dimerization in preventing unregulated gene expression. , 1999, Molecular cell.

[17]  Stephen K. Burley,et al.  Co-crystal structure of TBP recognizing the minor groove of a TATA element , 1993, Nature.

[18]  K. Lumb,et al.  Reevaluation of transcriptional regulation by TATA-binding protein oligomerization: predominance of monomers. , 2000, Biochemistry.

[19]  M. Portnoy,et al.  Dimer dissociation and thermosensitivity kinetics of the Saccharomyces cerevisiae and human TATA binding proteins. , 1999, Biochemistry.

[20]  A. Hoffmann,et al.  Crystal structure of TFIID TATA-box binding protein , 1992, Nature.

[21]  T Lagrange,et al.  The general transcription factors of RNA polymerase II. , 1996, Genes & development.

[22]  Russell P. Darst,et al.  Mot1 Regulates the DNA Binding Activity of Free TATA-binding Protein in an ATP-dependent Manner* , 2003, The Journal of Biological Chemistry.

[23]  B. Pugh,et al.  Dimerization of TFIID When Not Bound to DNA , 1996, Science.

[24]  B. Pugh,et al.  Slow dimer dissociation of the TATA binding protein dictates the kinetics of DNA binding. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[25]  B. Pugh,et al.  Evidence for Functional Binding and Stable Sliding of the TATA Binding Protein on Nonspecific DNA (*) , 1995, The Journal of Biological Chemistry.

[26]  J. Beechem,et al.  Yeast TATA binding protein interaction with DNA: fluorescence determination of oligomeric state, equilibrium binding, on-rate, and dissociation kinetics. , 1995, Biochemistry.

[27]  B. Cullen,et al.  A yeast TATA-binding protein mutant that selectively enhances gene expression from weak RNA polymerase II promoters , 1997, Molecular and cellular biology.