TY - JOUR
T1 - Dynamic interplay of TFIIA, TBP and TATA DNA
AU - Weideman, Christian A.
AU - Netter, Robert C.
AU - Benjamin, Lawrence R.
AU - McAllister, John J.
AU - Schmiedekamp, Lumelle A.
AU - Coleman, Robert A.
AU - Pugh, B. Franklin
N1 - Funding Information:
This work was supported by grants from the National Institutes of Health (GM47855) and the Leukemia Society of America. We thank D. Reinberg for the TFIIA expression plasmids. C.A.W. and R.C.N. contributed equally to this work.
PY - 1997/8/8
Y1 - 1997/8/8
N2 - The TATA binding protein (TBP) binds to the -30 region of eukaryotic and archaea promoters, where it assembles a transcription complex. For those genes transcribed by RNA polymerase II, transcription factor TFIIA binds TBP and positively regulates its activity, including enhancing TBP/ TATA interactions. Since little is known about the dynamic interplay among TFIIA, TBP and DNA, we set out to examine the stability of these interactions. Using the nitrocellulose filter binding assay, the k(off) of recombinant human TBP from TATA and non-specific DNA was determined to be 5.5(± 0.1) x 10-5 s-1 (t(1/2) = 210 minutes) and 5.8(± 0.1) x 10-4 s-1 (t(1/2) = 20 minutes), respectively. TFIIA/TBP complexes, containing either HeLa-derived or recombinant human TFIIA, possessed a nearly tenfold lower k(off) when bound to TATA. Interactions of TFIIA with DNA upstream of the TATA box did not appear to play a major role in stabilizing TBP/TATA interactions. Instead, the upstream DNA contacts appeared to be important for stabilizing the association of TFIIA with the TBP/TATA complex as measured in electrophoretic mobility shift assays: k(off) of TFIIA decreased from 1.4(± 0.1)x 10-3 s-1 (t(1/2) = eight minutes) to 2.4(± 0.2) x 10-4 s-1 (t(1/2) = 49 minutes) when upstream DNA contacts were allowed. The stability of TFIIA/TBP interactions was measured using a rapid 'pull-down' assay, which employed-nickel agarose and polyhistidine-tagged TFIIA. In the absence of DNA, TFIIA dissociated from TBP with a k(off) = 4.9(± 0.6) x 10-3 s-1 (t(1/2) = 2.4 minutes), which varied with solution conditions.
AB - The TATA binding protein (TBP) binds to the -30 region of eukaryotic and archaea promoters, where it assembles a transcription complex. For those genes transcribed by RNA polymerase II, transcription factor TFIIA binds TBP and positively regulates its activity, including enhancing TBP/ TATA interactions. Since little is known about the dynamic interplay among TFIIA, TBP and DNA, we set out to examine the stability of these interactions. Using the nitrocellulose filter binding assay, the k(off) of recombinant human TBP from TATA and non-specific DNA was determined to be 5.5(± 0.1) x 10-5 s-1 (t(1/2) = 210 minutes) and 5.8(± 0.1) x 10-4 s-1 (t(1/2) = 20 minutes), respectively. TFIIA/TBP complexes, containing either HeLa-derived or recombinant human TFIIA, possessed a nearly tenfold lower k(off) when bound to TATA. Interactions of TFIIA with DNA upstream of the TATA box did not appear to play a major role in stabilizing TBP/TATA interactions. Instead, the upstream DNA contacts appeared to be important for stabilizing the association of TFIIA with the TBP/TATA complex as measured in electrophoretic mobility shift assays: k(off) of TFIIA decreased from 1.4(± 0.1)x 10-3 s-1 (t(1/2) = eight minutes) to 2.4(± 0.2) x 10-4 s-1 (t(1/2) = 49 minutes) when upstream DNA contacts were allowed. The stability of TFIIA/TBP interactions was measured using a rapid 'pull-down' assay, which employed-nickel agarose and polyhistidine-tagged TFIIA. In the absence of DNA, TFIIA dissociated from TBP with a k(off) = 4.9(± 0.6) x 10-3 s-1 (t(1/2) = 2.4 minutes), which varied with solution conditions.
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U2 - 10.1006/jmbi.1997.1152
DO - 10.1006/jmbi.1997.1152
M3 - Article
C2 - 9300055
AN - SCOPUS:0031559556
SN - 0022-2836
VL - 271
SP - 61
EP - 75
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -