DNA and protein footprinting analysis of the modulation of DNA binding by the N-terminal domain of the Saccharomyces cerevisiae TATA binding protein.
Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by "protein footprinting" with hydroxyl radical (*OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.
Center for Synchrotron Biosciences, National Synchrotron Light Source, Brookhaven National Laboratory, Upton, New York 11973, USA.
Sayan
S
Gupta
Huiyong
H
Cheng
A K M M
AK
Mollah
Elizabeth
E
Jamison
Stephanie
S
Morris
Mark R
MR
Chance
Sergei
S
Khrapunov
Michael
M
Brenowitz
D020816:Amino Acid Motifs; D001665:Binding Sites; D002592:Cetomacrogol / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007266:Inhibition, Psychological; D008958:Models, Molecular; D023302:Protein Footprinting; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry / Q000378:metabolism; D013501:Surface-Active Agents / Q000737:chemistry; D035182:TATA Box Binding Protein-Like Proteins / Q000378:metabolism; D035381:TATA-Binding Protein Associated Factors / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D013501:Surface-Active Agents; D035182:TATA Box Binding Protein-Like Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D002592:Cetomacrogol; D004247:DNA