Genetic and biochemical analyses of yeast TATA-binding protein mutants

We have taken a combined genetic and biochemical approach to study TATA-binding protein (TBP) structure-function relationships. Using site-directed mutagenesis coupled with a screen for conditional lethal growth, we have isolated a number of temperature-sensitive TBP alleles in the region of amino a...

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Bibliographic Details
Published in:The Journal of biological chemistry 1993-03, Vol.268 (7), p.5005-5013
Main Authors: POON, D, KNITTLE, R. A, SABELKO, K. A, YAMAMOTO, T, HORIKOSHI, M, ROEDER, R. G, WEIL, P. A
Format: Article
Language:English
Subjects:
Alleles
Amino Acids - metabolism
Analytical, structural and metabolic biochemistry
Base Sequence
Binding and carrier proteins
Biological and medical sciences
DNA, Fungal - metabolism
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Fundamental and applied biological sciences. Psychology
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Binding
Proteins
RNA Polymerase II - metabolism
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - isolation & purification
Saccharomyces cerevisiae - metabolism
Substrate Specificity
TATA Box
TATA-Box Binding Protein
Temperature
Transcription Factors - genetics
Transcription Factors - metabolism
Transcription, Genetic
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Summary:We have taken a combined genetic and biochemical approach to study TATA-binding protein (TBP) structure-function relationships. Using site-directed mutagenesis coupled with a screen for conditional lethal growth, we have isolated a number of temperature-sensitive TBP alleles in the region of amino acid positions 188, 189, and 190. Conditional growth is not a result of increased TBP turnover as most of the mutant proteins are stable in vivo as evidenced by immunoblot detection of TBP steady-state levels. DNA binding assays reveal that mutations at position 188 do not affect DNA binding activity of these mutants, even at high temperatures. Utilizing whole cell extracts which contain mutant TBPs in in vitro transcription experiments, we confirm that TBP is required for transcription by all three nuclear polymerases. However, certain of our TBP mutants are only compromised for RNA polymerase II transcription.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)53495-5