Kinetic analysis of yeast TFIID-TATA box complex formation suggests a multi-step pathway.
The eukaryotic transcription factor TFIID recognizes and binds a promoter sequence element called the TATA box. We have analyzed the interaction of yeast TFIID with the consensus TATA box sequence of the adenovirus major late promoter. To facilitate this detailed characterization, we developed a method for obtaining quantitative information from a gel retardation (bandshift) assay, allowing measurement of the rate and extent of TFIID-TATA box complex formation. Using this assay and DNase I protection assays, we determined that the association rate constant for TFIID binding to the major late promoter was too low to be consistent with a simple diffusion-limited association, suggesting that the binding proceeds by a multi-step pathway. Furthermore, we found that the slow rate of TFIID binding reported by other research groups was not the consequence of a rate-limiting conformational change, as has been previously suggested. Instead, we observed that the formation of a stable TFIID-TATA box complex was relatively rapid (complete in less than 1 min) at saturating concentrations of TFIID. We have proposed a two-step pathway consistent with the observed kinetics and have considered the possible contributions of each step to the overall rate of TFIID binding. This study lays the groundwork for a systematic characterization of the interaction of TFIID with additional TATA box sequences, including an experimental test of the possibility that different steps in the binding reaction are rate-limiting for different promoters.
Hoopes
B C
BC
Institute of Molecular Biology, University of Oregon, Eugene 97403.
LeBlanc
J F
JF
Hawley
D K
DK
D016172:DNA Fingerprinting; D004268:DNA-Binding Proteins / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016385:TATA Box; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins
D004268:DNA-Binding Proteins; C051096:MLTF protein, adenovirus; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D014764:Viral Proteins