RPC10 encodes a new mini subunit shared by yeast nuclear RNA polymerases

Abstract

Yeast RNA polymerases A, B, and C share five small subunits, two of which, ABC10 alpha and ABC10 beta, comigrate on SDS polyacrylamide gels. The gene encoding ABC10 alpha, RPC10, was isolated based on microsequence data. RPC10 is a single copy gene localized on chromosome VIII. It codes for a very basic protein of only 70 amino acids, which contains a zinc binding domain of the form CX2CX13CX2C. Deletion of its gene indicated that, despite its very small size, the ABC10 alpha subunit is essential for yeast cell viability. ABC10 alpha and ABC10 beta have little sequence similarity.

Figure 1

Nucleotide sequence of RPC10 coding and flanking regions. The uninterrupted open reading frame of 210 bp encodes a sequence of 70 amino acids which contains the two sequenced tryptic peptides (underlined). The four cysteines of the putative zinc binding domain are circled. The basic heptapeptide discussed in the text is underlined (dotted line). In the upstream region, a consensus ABF1 binding site and a T-rich element are boxed. The position of the Nru I and EcoN I restriction sites used for gene disruption are indicated.

Figure 2

RPC10 is a single copy gene. Yeast genomic DNA from YNN281 was digested with the indicated restriction enzymes (E, EcoR I; H, Hind III; X, Xba I; S, Stu I; En, EcoN I), electrophoresed on a 0.7% agarose gel, transferred onto a nitrocellulose membrane, and probed with the ³²P-end labeled 30-mer oligonucleotide as described in Materials and Methods. The size of marker DNA fragments is given in kilobases.

Figure 3

Chromosomal localization of RPC10. Chromosomal DNA of YNN295 yeast strain was separated by pulse field electrophoresis and stained with ethidium bromide (EB). After transfer onto a nitrocellulose membrane, the blotted DNA was hybridized with the Nru I-EcoN I ³²P-labeled fragment. The labeled chromosomal DNA band was identified by auto radiography (³²P). The probe hybridized strongly to chromosome VIII (arrow). The dotted arrow points to the faint hybridization signal on chromosome IV.

Figure 4

Chromosomal disruption of RPC10. A. Restriction map of RPC10 chromosomal locus and replacement of the Nru I-EcoN I internal fragment by HIS3 gene. Restriction sites are: H, Hind III; E, EcoR I; N, Nru I; En, EcoN I; B, BamH I. The arrows indicate the size of relevant restriction fragments. B. Southern analysis of the gene disruption. The diploid strain CMY214 (his⁻) was transformed with the 3.7 kb EcoR I fragment containing RPC10 disrupted by the HIS3 gene. The genomic DNA of several HIS⁺ transformants was isolated, restricted with EcoR I, and subjected to Southern analysis by hybridization with the 2.1 kb EcoR I fragment. Lanes 1, 2, and 3 show the Southern blot from three HIS⁺ transformants; wt, control hybridization with DNA from the original CMY214 strain. C. Tetrad analysis from one HIS⁺ disrupted diploid transformant containing one wild-type and one disrupted copy of RPC10. All dissected tetrads contained only two viable spores on YPD medium which were always his⁻.