Sequence analysis of C17. The yeast C17 protein sequence (GenBank accession no. Z49286) was aligned with different orthologs, as indicated. C. al., Candida albicans protein of unknown function, unfinished fragment of complete genome, Stanford database; C. el., Caenorhabditis elegans protein of unknown function (accession no. CAA87050); H. sap., Homo sapiens CGRP receptor component protein (accession no. AF073792); C. porc., Cavia porcellus CGRP receptor component protein (accession no. U50188); M. mus., Mus musculus CGRP receptor component protein (accession no. AF028242); G. gal., Gallus gallus protein of unknown function, partial sequence (accession no. D26313). The complete sequences are shown except for C. elegans and G. gallus proteins, were residues that showed no homology with C17 are not included. Amino acid positions are indicated; residues identical in five sequences are boxed, and conserved substitutions are shaded.

Interaction of C17 with wild-type or mutant TFIIIB70 proteins. The two-hybrid system was used to investigate protein-protein interactions between C17 and TFIIIB70. The ORF of C17 was fused in frame with the GAL4 DNA-binding domain (amino acids 1 to 147). Wild-type or mutant TFIIIB70 ORF was fused in frame with the GAL4 activation domain (amino acids 768 to 881). Numbers in parentheses indicate the TFIIIB70 amino acids present in the fusion protein. TFIIIB70 motifs homologous to TFIIB are indicated. Transcription activation of the lacZ reporter gene was assayed by growing the transformed cells on selective medium and overlaying with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) agar. White (−) and blue (+) coloration of cell patches on X-Gal plates is indicated.

A 17-kDa polypeptide in RNA polymerase III from S. cerevisiae. Pol III purified as described in Materials and Methods was analyzed by electrophoresis under denaturing conditions in an SDS–12% polyacrylamide gel. Proteins were then stained by Coomassie blue. Lane 1, Pol III from a wild-type strain; lane 2, Pol III from a strain in which yABC23 is replaced by its human counterpart hABC23.

In vivo synthesis of small RNA species after repression of RPC17 gene expression. Strain YMLF2 harboring plasmid pCMc17, which allows the modulation of RPC17 expression by changing doxycycline concentration in the growth medium, was grown at 30°C with (+) or without (−) the addition of doxycycline (5 μg/ml). Tritiated uracil incorporation was allowed for 10 min after 6 or 12 h of growth, as indicated. RNA species were extracted and analyzed by electrophoresis on a 7 M urea gel (6% polyacrylamide), using equal amounts of RNA (6 μg per lane). (A) Ethidium bromide staining of RNA; (B) autoradiography of the dried gel shown in panel A.

Coimmunoprecipitation assay of C17 and N-terminally truncated TFIIIB70 fragments. Crude extracts (60 μg) with (+) or without (−) T7-tagged C17 or His₆-tagged rTFIIIB70 were preincubated for 45 min at 25°C and then mixed with magnetic beads coated with anti-T7 antibodies. Crude extracts (60 μg) with or without rTFIIIB70 were also preincubated with purified recombinant TBP (240 ng), as indicated, and then mixed with magnetic beads coated with anti-TBP antibodies. The beads were washed, and bound proteins were eluted by boiling the beads in loading buffer. (A) Western blot analysis. The input and bound proteins were analyzed by Western blotting using anti-T7, antipentahistidine, or anti-TFIIIB70 antibodies, as indicated. Asterisks indicate positions of immunoglobulin heavy and light chains; positions and molecular masses (in kilodaltons) of marker bands are indicated at the left. The molecular masses (in kilodaltons) of full-length and truncated rTFIIIB70 polypeptides present in crude extracts are shown. The position of rC17, migrating at 23 kDa, is indicated. IP, immunoprecipitation. (B) Quantification of rTFIIIB70 immune complexes. The immune complexes shown in panel A revealed with anti-TFIIIB70 antibodies (lanes 2, 6, and 10) were quantified with ImageQuant software. The results for each component of rTFIIIB70 are shown as percentage of total immune complexes.