Abstract
Transcription by RNA polymerase III (pol III) in yeast requires the assembly of an initiation complex comprising the TATA-binding protein (TBP), a 90-kDa polypeptide (TFIIIB90), and a 70-kDa polypeptide (TFIIIB70). TFIIIB70 interacts with TBP, a unique pol III subunit, C34, and the 131-kDa subunit of the pol III-specific complex, TFIIIC. TFIIIB70 was expressed in Escherichia coli and purified to homogeneity. The specific transcription activity of rTFIIIB70 is 22-58% that of the native yeast and in vitro synthesized factor. However, only a small fraction (0.07-0.32%) of the TFIIIB70 from these sources results in the synthesis of full-length RNA. The data suggest that TFIIIB70 function may be limited by an unfavorable recruitment equilibrium into the preinitiation complex. Quantitative DNase I "footprint" titrations of yeast TBP to the adenovirus major late promoter were conducted at a series of constant TFIIIB70 concentrations. A value of -0.7 ± 0.2 kcal/mol was determined for the cooperative free energy of formation of the TBP-TFIIIB70-DNA complex at concentrations of TFIIIB70 sufficient to partition all of the binding cooperativity to the TBP binding isotherm. A Kd of 44 ± 23 nM characterizes the TFIIIB70 concentration dependence of the TBP-TFIIIB70 cooperativity. The relationship δlog K/δlog (TFIIIB70) is consistent with the linkage of a single molecule of TFIIIB70 with the TBP-promoter binding reaction.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 32695-32701 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 271 |
| Issue number | 51 |
| State | Published - 1996 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Expression and purification of the RNA polymerase III transcription specificity factor IIIB70 from Saccharomyces cerevisiae and its cooperative binding with TATA-binding protein. / Librizzi, Monett D.; Moir, Robyn D.; Brenowitz, Michael D.; Willis, Ian M.
In: Journal of Biological Chemistry, Vol. 271, No. 51, 1996, p. 32695-32701.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Expression and purification of the RNA polymerase III transcription specificity factor IIIB70 from Saccharomyces cerevisiae and its cooperative binding with TATA-binding protein
AU - Librizzi, Monett D.
AU - Moir, Robyn D.
AU - Brenowitz, Michael D.
AU - Willis, Ian M.
PY - 1996
Y1 - 1996
N2 - Transcription by RNA polymerase III (pol III) in yeast requires the assembly of an initiation complex comprising the TATA-binding protein (TBP), a 90-kDa polypeptide (TFIIIB90), and a 70-kDa polypeptide (TFIIIB70). TFIIIB70 interacts with TBP, a unique pol III subunit, C34, and the 131-kDa subunit of the pol III-specific complex, TFIIIC. TFIIIB70 was expressed in Escherichia coli and purified to homogeneity. The specific transcription activity of rTFIIIB70 is 22-58% that of the native yeast and in vitro synthesized factor. However, only a small fraction (0.07-0.32%) of the TFIIIB70 from these sources results in the synthesis of full-length RNA. The data suggest that TFIIIB70 function may be limited by an unfavorable recruitment equilibrium into the preinitiation complex. Quantitative DNase I "footprint" titrations of yeast TBP to the adenovirus major late promoter were conducted at a series of constant TFIIIB70 concentrations. A value of -0.7 ± 0.2 kcal/mol was determined for the cooperative free energy of formation of the TBP-TFIIIB70-DNA complex at concentrations of TFIIIB70 sufficient to partition all of the binding cooperativity to the TBP binding isotherm. A Kd of 44 ± 23 nM characterizes the TFIIIB70 concentration dependence of the TBP-TFIIIB70 cooperativity. The relationship δlog K/δlog (TFIIIB70) is consistent with the linkage of a single molecule of TFIIIB70 with the TBP-promoter binding reaction.
AB - Transcription by RNA polymerase III (pol III) in yeast requires the assembly of an initiation complex comprising the TATA-binding protein (TBP), a 90-kDa polypeptide (TFIIIB90), and a 70-kDa polypeptide (TFIIIB70). TFIIIB70 interacts with TBP, a unique pol III subunit, C34, and the 131-kDa subunit of the pol III-specific complex, TFIIIC. TFIIIB70 was expressed in Escherichia coli and purified to homogeneity. The specific transcription activity of rTFIIIB70 is 22-58% that of the native yeast and in vitro synthesized factor. However, only a small fraction (0.07-0.32%) of the TFIIIB70 from these sources results in the synthesis of full-length RNA. The data suggest that TFIIIB70 function may be limited by an unfavorable recruitment equilibrium into the preinitiation complex. Quantitative DNase I "footprint" titrations of yeast TBP to the adenovirus major late promoter were conducted at a series of constant TFIIIB70 concentrations. A value of -0.7 ± 0.2 kcal/mol was determined for the cooperative free energy of formation of the TBP-TFIIIB70-DNA complex at concentrations of TFIIIB70 sufficient to partition all of the binding cooperativity to the TBP binding isotherm. A Kd of 44 ± 23 nM characterizes the TFIIIB70 concentration dependence of the TBP-TFIIIB70 cooperativity. The relationship δlog K/δlog (TFIIIB70) is consistent with the linkage of a single molecule of TFIIIB70 with the TBP-promoter binding reaction.
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M3 - Article
C2 - 8955101
AN - SCOPUS:0030478516
VL - 271
SP - 32695
EP - 32701
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 51
ER -