RNA Polymerase II Subunit RPBlO Is Essential for Yeast Cell Viability*

The Saccharomyces cerevisiae gene encoding the smallest RNA polymerase II subunit, RPB10, was isolated and sequenced. The gene for this subunit is present in single copy and maps to chromosome XV, where two other yeast RNA polymerase II subunits, RPB2 and RPB8, reside. The RPB10 sequence predicts a protein only 46 amino acids in length with a molecular mass of 5400 daltons. Sporulation and tetrad analysis of diploid cells containing one copy of the RPB10 gene and one copy of HIS3 in place of the RPB10 gene revealed that the RPB10 subunit is essential for viability.

The Saccharomyces cerevisiae gene encoding the smallest RNA polymerase II subunit, RPBlO, was isolated and sequenced.
The gene for this subunit is present in single copy and maps to chromosome XV, where two other yeast RNA polymerase II subunits, RPB2 and RPB8, reside.
The RPBlO sequence predicts a protein only 46 amino acids in length with a molecular mass of 5400 daltons.
Sporulation and tetrad analysis of diploid cells containing one copy of the RPBlO gene and one copy of HIS3 in place of the RPBlO gene revealed that the RPBlO subunit is essential for viability.
To understand better the structure and function of eukaryotic RNA polymerases, the genes that encode S. cereuisiue RNA polymerase II subunits are being isolated and used to examine the roles of subunits in mRNA transcription. Thus far, the genes for the yeast RNA polymerase II subunit RPBl (Young and Davis, 1983;Ingles et al., 1984;Allison et al., 1985); RPBP (Sweetser et al., 1987); RPB3 (Kolodziej and Young, 1989); RPB4 (Woychik and Young, 1989); and RPBS, RPBG, and RPB8  have been isolated and sequenced. In this paper we describe the isolation and sequence of the smallest RNA polymerase II subunit, RPBlO, as well as data that demonstrate that it is essential for cell viability.  was constructed based on codon usage data combined with substitution of deoxyinosine (I) residues at positions where no sequence was available.
Transfer of bacterial colonies to filter membranes was done by placing a dry nitrocellulose circle over the colonies on the surface of a chilled plate (4 "C) for 1 min. The filter was removed and left to air dry for 10 min. Lysis of the cells and denaturation of nucleic acids were achieved by-placing the filters in the autoclave for 2 min on the dry cycle. After baking in an 80 "C vacuum oven for l-2 h, the filters were washed for 30 min in 2 x SSC (Duby et al., 1989) ' Allison et al. (1985). . 'Mann et al. (1987). d Pati and Weismann (1989). To construct a plasmid library enriched in RPBlO DNA, S. cereuisicze genomic DNA was digested with P&I, and DNA fragments in the range of 2-4 kb were gel purified and ligated to P&I-cut pBluescript KS+ DNA. Escherichia coli cells were transformed with this DNA, the plasmid library was screened with the RPBlO oligonucleotide probe, and signal-producing colonies were isolated. One positive clone was obtained from approximately 800 colonies. Plasmid prepared from this clone contained a 3.0-kb PstI DNA fragment. Southern analysis of multiple restriction digests of this insert DNA using the RPBlO 80-mer oligonucleotide as a probe revealed that the DNA of interest was included within a 1.5-kb PstI/SphI fragment.
The DNA sequence of the 1.5-kb PstI/SphI fragment was determined, and the sequence of the RPBlO amino-terminal peptide was found within one of the open reading frames. The position of the RPBlO coding sequence (Fig. 1) relative to the restriction map was deduced from the sequence of the PstI/ SphI fragment. The RPBlO sequence (Fig. 2) predicts a protein of 46 amino acids with a molecular mass of 5400 Da, considerably lower than the apparent molecular mass of 10 kDa obtained from SDS-PAGE.
There is considerable error associated with the molecular mass estimate obtained from original SDS-PAGE experiments because the polyacrylamide gels used in these estimates were designed to resolve a very broad range of protein sizes (lo-220 kDa). To assess better the SDS-PAGE mobility of RPBlO, we ran purified S. cereuisiue RNA polymerase II adjacent to low molecular mass markers on a high percentage (12.5%) SDS-polyacrylamide gel. This experiment revealed that RPBlO actually runs as a 6-kDa protein, very close its actual molecular mass of 5.4 kDa (not shown).
Analysis of the RPBlO coding sequence revealed that the gene encodes a very basic protein, with a p1 of 9.6. A computer search of conventional databases did not reveal the existence of any protein sequences significantly similar to RPBIO. However, RPBlO has a potential metal-binding domain, C-X-X-C-G, where X is any amino acid. Previous studies of the ironbinding protein rubredoxin revealed that two of these sequence motifs are involved in binding one Fe'+ (Adman et al., 1975). Although this metal-binding sequence motif occurs rarely among proteins in the database, it is found in the amino terminus of eukaryotic and prokaryotic RNA polymerases (Fig. 3).

Copy Number and Chromosomal Location of Common Subunit Genes-Southern
blots containing immobilized restriction digests of S. cereuisiae genomic DNA were probed with RPBlO DNA fragments at moderate stringency as described under "Experimental Procedures" (Fig. 4). The pattern of hybridization, in which only a single band producing a strong signal was observed, indicated that RPBlO is a single copy gene in haploid yeast. The pattern of hybridization obtained with the RPBlO DNA probe did not change over a range of hybridization and wash conditions and was the same as that obtained with the oligonucleotide probe used for gene isolation and characterization.
The RPBlO gene was localized to chromosome XV by probing a Southern blot containing S. cereuisiae chromosome separated by pulsed field electrophoresis with gene-specific DNA fragments (Fig. 5). Two other RNA polymerase II subunits, RPB2 and RPB8, have also been mapped to chromosome XV (Scafe et al., 1990;Woychik et al., 1990). Examination of predicted amino acid sequences verified that RPBlO is not a degradation product of either gene. It is not yet clear whether these three genes are linked, but the DNA sequences that flank the gene do not overlap. The other five RNA polymerase II subunit genes that have been isolated reside on separate chromosomes (Table I).
RPBlO Is Essential for Cell Viability-Most, but not all, of the RNA polymerase subunit genes studied thus far are essential for yeast cell viability (Nonet et al., 1987;Kolodziej and Young, 1989;Woychik and Young, 1989;Woychik et al., 1990). To determine whether RPBlO is essential for cell viability, the gene was replaced with the yeast nutritional marker HIs3. The entire protein coding region of RPBlO was first deleted and replaced with two unique restriction sites using oligonucleotide-directed mutagenesis (Kunkel, 1985). A HIS3 DNA fragment was inserted into the newly created restriction sites to produce the rpblOAl::HIS3 allele.
One chromosomal copy of the gene in diploid yeast cells was replaced using the method of Rothstein (1983). This method relies on homologous recombination of RPBIO-flanking DNA with the chromosomal DNA, resulting in the replacement of Yeast RNA Polymer-cue II Gene RPBlO one of the chromosomal copies of the subunit gene with a selectable marker. The diploid cells obtained by this approach have one chromosome with a wild-type RNA polymerase subunit gene and one chromosome with a deletion allele. Tetrad analysis of the sporulation products of these diploid cells revealed that the deletion of RPBlO produces nonviable haploid cells (Table II). Therefore, RPBlO is essential for cell viability.

DISCUSSION
RPBlO is the smallest of the 10 RNA polymerase II subunits, consisting of 46 amino acids with a molecular mass of 5.4 kDa. It is a single copy gene located on chromosome XV. Despite its size, the RPBlO subunit is required for cell viability.
Eight of the 10 RNA polymerase II subunits have now been identified ( Table I). The two largest subunits, RPBl and RPBB, are homologs of the bacterial RNA polymerase subunits, p' and p, respectively. The two large subunits of eukaryotic and prokaryotic RNA polymerases can bind DNA and nucleoside triphosphate substrates and are thought to contain the catalytic site (Chamberlin, 1982;Yura and Ishihama, 1979;Carroll and Stollar, 1983;Cho and Kimball, 1982;Riva et al., 1987). Amino acid sequence similarity between RPB3 and the bacterial a-subunit coupled with the fact that the behavior of RPB3 assembly mutants parallels that of a-subunit assembly mutants suggests that RPB3 is an cY-homolog.' The other RNA polymerase II subunits do not exhibit extensive sequence similarity to other prokaryotic RNA polymerase subunits. All but one of the eight yeast RNA polymerase II subunits isolated to date are essential for cell viability.
The exception, RPB4, is not required for viability but is essential for high and low temperature yeast cell growth. RPBlO is among the RNA polymerase II polypeptides that play a critical role in transcription.
The function of RPBlO is unclear, but the constraints of its size would appear to limit its functional potential. It seems likely that RPBlO plays either a structural role or acts as an accessory to the function of other subunits. Precisely defining the function of RPBlO will require further molecular genetic approaches.