Abstract
The yeast transcription factor IIIC (TFIIIC) is organized in two distinct multisubunit domains, tauA and tauB, that are respectively responsible for TFIIIB assembly and stable anchoring of TFIIIC on the B block of tRNA genes. Surprisingly, we found that the removal of tauA by mild proteolysis stabilizes the residual tauB.DNA complexes at high temperatures. Focusing on the well conserved tau95 subunit that belongs to the tauA domain, we found that the tau95-E447K mutation has long distance effects on the stability of TFIIIC.DNA complexes and start site selection. Mutant TFIIIC.DNA complexes presented a shift in their 5' border, generated slow-migrating TFIIIB.DNA complexes upon stripping TFIIIC by heparin or heat treatment, and allowed initiation at downstream sites. In addition, mutant TFIIIC.DNA complexes were highly unstable at high temperatures. Coimmunoprecipitation experiments indicated that tau95 participates in the interconnection of tauA with tauB via its contacts with tau138 and tau91 polypeptides. The results suggest that tau95 serves as a scaffold critical for tauA.DNA spatial configuration and tauB.DNA stability.
Highlights
TFIIIC1 is a multisubunit DNA binding factor that serves as a dynamic platform to assemble pre-initiation complexes on class III genes
Focusing on the well conserved 95 subunit that belongs to the A domain, we found that the 95-E447K mutation has long distance effects on the stability of TFIIIC1⁄7DNA complexes and start site selection
A Affects TFIIIC1⁄7DNA Binding—As the above observations could not account for the strong thermosensitivity and the drop of in vivo tRNA synthesis occurring in the YSJ1-E447K strain, we looked for other major defects in mutant transcription factor IIIC (TFIIIC). 95 is thought to be involved in block A binding (8, 11), but the overall DNA affinity of TFIIIC is predominantly governed by the interaction of B domain with the B block (5)
Summary
DNA Constructions and TFC1 Random Mutagenesis—The 2.8-kb XhoI/NotI fragment from plasmid YcpCS7 (CEN, URA, wt HA-TFC1) (27), containing the promoter region and a modified open reading frame of TFC1 allowing addition of an HA tag sequence after the initiation codon, was cloned into the polylinker region of plasmid pRS314 (28) creating pYSJ1 (CEN, TRP, wt HA-TFC1). A 32P-labeled DNA fragment (3–10 fmol; 4,000 –10,000 cpm) carrying the SUP4 tRNATyr (345 bp) gene was incubated for 10 min at 25 °C in a reaction mixture containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10% glycerol, 2 mg/ml bovine serum albumin (Sigma), DNA competitor, TFIIIC (MonoQ fraction) at a final KCl concentration of 120 mM. The apparent dissociation constants (Kapp) of wild type and mutant TFIIIC-DNA complexes were determined, using the above binding conditions as described (4, 29). Pellets were resuspended in 10 l of denaturing loading buffer (90% (v/v) formamide, 10 mM Tris-HCl (pH 8), 1 mM EDTA, bromphenol blue, and xylene cyanol), heated for 5 min at 90 °C, and loaded onto a 7% (w/v) polyacrylamide-urea sequencing gel in parallel with 32P-labeled DNA kb ladder (Invitrogen). Immunopurified proteins were eluted by heating for 3 min at 95 °C in SDS loading buffer and analyzed by SDS-PAGE on a 8% polyacrylamide gel and revealed by Western blotting with 12CA5 anti-HA, M2 anti-FLAG (Sigma), or anti--91 antibodies, using an Amersham enhanced chemiluminescence kit
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