Transcription Factor TFIID
Postdoctoral Research at LBNL
Current research in collaboration with the lab of Robert Tjian is to obtain the three-dimensional structure of the transcription factor TFIID using single particle reconstruction of images obtained using transmission electron microscopy. TFIID is localized within the nucleus of the cell and, along with other basal transcription factors, is primarily responsible for showing RNA polymerase the start of a transcription site by binding to the DNA TATA box upstream of a gene. TFIID recognizes the TATA sequence through a special protein called the TATA binding protein (TBP) The determination of the structure of TFIID pictured below allows us to answer important questions about its function.
Three-dimensional reconstruction of human TFIID at 35 Anstroms resolution. (A) and (B) are front and back views, respectively, showing the overall horseshoe structure of TFIID. The three lobes have been designated A, B, and C. (C) to (E) are side views obtained by turning the front view in (A) out of the paper by 90 degrees, then rotating about a horizontal axis through the center of the cavity. The "V" shaped clefts between lobes are indicated by arrows.
Below is a series of views of the 3D structure rotated about the horizontal and vertical tilt axes. TFIID has a horseshoe or "crescent" shape with a central cavity. Improved resolution to 30 Anstroms (shown in the second movie below) reveals even more detailed features. Continued improvement of resolution using techniques such as cryoEM should improve resolution to below 15 Anstroms.
3D reconstructions of TFIID at 35 and 30 Angstroms resolution.
We were interested in determining the DNA binding site within the TFIID structure. Three independent reconstructions of TFIID provided us with this information. Since two other transcription factors were known to bind to opposite sides of TBP, TFIIA and TFIIB, we calculated the structure of TFIID with these bound factors and later with an antibody raised against TBP (shown in the figure below). Taken together, these data suggest that TBP resides at the top of the central cavity of TFIID.
Position of TFIIB and TFIIA on the TFIID structure and mapping of the TBP. The blue mesh corresponds to the holo-TFIID. The green mesh corresponds to the density difference between the holo-TFIID and the TFIID-IIB complex. The magenta mesh shows the density difference between the holo-TFIID and the trimeric complex TFIID-IIA. The yellow mesh shows the density difference between the holo-TFIID and TFIID bound to the TBP antibody.
Thanks goes to Carla Inouye and Andreas Ladurner of the Tjian laboratories who isolated the TFIID used in these studies. Thanks also to William Nicholson for getting me started with SPIDER. Ken Downing gave me the necessary help with microscopy to succeed. Bob Glaeser always had an idea up his sleeve. Ed Hoch turned me on to animated gifs. I also acknowledge Pawel Penczek for stimulating discussions concerning 3D variance and single particle reconstruction.
by Frank Andel III
©1999
