Visualization of TBP oligomers binding and bending the HIV-1 and adeno promoters.
The binding of the 28 kDa yeast TATA binding protein (yTBP) to the HIV and adeno major late promoters has been examined by electron microscopy (EM). Three different EM preparative methods were employed: direct mounting and shadowcasting of fixed samples, cryofixation and freeze-drying followed by shadowcasting, and negative staining of unfixed samples. Excellent agreement among the three methods was obtained. With ten yTBP monomers/DNA fragment, up to 25% of the DNA molecules contained easily distinguished protein particles at the TATA box and, less frequently, smaller particles were observed. Non-specific binding to DNA ends was common. The mass of the easily distinguished particles measured 63(+/- 5) kDa (cryofixation and shadowcasting) and 48(+/- 6) kDa (negative staining) indicating TBP dimerization. With 22 and 44 yTBP monomers/DNA, yTBP polymerization produced DNA-protein rods 9 nm wide and 20 to 30 nm long, frequently with two DNA strands exiting one end. Bending analysis revealed that yTBP dimers bend the DNA about the TATA box by 80 to 90 degrees. Although these protein ratios are relatively high, the structures formed demonstrate the propensity of yTBP to engage in protein-protein interactions.
Griffith
J D
JD
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295.
Makhov
A
A
Zawel
L
L
Reinberg
D
D
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D001704:Biopolymers; D015925:Cryopreservation; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D005612:Freeze Drying; D015497:HIV-1 / Q000235:genetics*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012441:Saccharomyces cerevisiae; D013194:Staining and Labeling; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure
D001704:Biopolymers; D004268:DNA-Binding Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors