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.