Initiation of enzymatic DNA synthesis by yeast RNA polymerase I.

In vitro DNA synthesis by yeast DNA polymerase I can be initiated by partially purified yeast RNA polymerases in the presence or absence of rNTPs. Homogeneous yeast RNA polymerase I initiates DNA synthesis by yeast DNA polymerase I on single-stranded DNA templates only in the presence of all four rNTPs. A protein capable of initiating enzymatic DNA synthesis on single-stranded DNA in the absence of rNTPs has also been separated from partially purified yeast RNA polymerase I fractions. Analysis of the RNA polymerase I initiated replication products of phage fd DNA on alkaline sucrose gradients showed noncovalent linkage between the newly synthesized DNA and the template. Isopycnic analyses of the ribonucleotide initiated fd DNA replication products demonstrated covalent linkage between the initiator RNA and newly synthesized DNA. Results from 32P-transfer experiments confirmed the covalent linkage between RNA and DNA chains and showed the presence of all four ribo- and deoxyribonucleotides at the RNA--DNA junctions. The ribonucleotide found most frequently at the RNA--DNA junction is uridylate and the purine deoxynucleotides occur more frequently than pyrimidine deoxynucleotides.     

Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.

The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986).     

Subunits of yeast RNA polymerase I involved in interactions with DNA and nucleotides.

Ovine mammary gland mRNAs were translated in a wheat-germ cell-free system in the presence of radioactive amino acids. Automated Edman degradation performed on α-lactalbumin isolated by immunoprecipitation from the mixture of radiolabelled lactoproteins showed the occurrence of an hydrophobic 19 residues long amino terminal extension. The pre-protein represents the primary translation product since the amino terminal methionyl residue was found to be donated by initiator $\text{Met-tRNA}{}_{\mathrm{i}}{}^{\mathrm{Met}}$. Comparison of the signals of ovine α-lactalbumin and hen's egg white lysozyme, two homologous proteins which are thought to be derived from a common ancestor, suggests that the signal region has evolved at least as rapidly as the remaining part of the polypeptide chain.     

Essential roles of the RNA polymerase I largest subunit and DNA topoisomerases in the formation of fission yeast nucleolus

A temperature-sensitive lethal mutant nuc1-632 of Schizosaccharomyces pombe shows marked reduction in macromolecular synthesis and a defective nuclear phenotype with an aberrant nucleolus, indicating a structural role of the nuc1+ gene product in nucleolar organization. We cloned the nuc1+ gene by transformation and found that it appears to encode the largest subunit of RNA polymerase I. We raised antisera against nuc1+ fusion polypeptides and detected a polypeptide (approximately 190 kD and 2 x 10(4) copies/cell) in the S. pombe nuclear fraction. By immunofluorescence microscopy, anti-nuc1+ antibody revealed intense staining at a particular nuclear domain previously defined as the nucleolus. The nucleolar immunofluorescence by anti-nuc1+ was faded in nuc1-632 at restrictive temperature and dramatically diminished in the absence of DNA topoisomerases I and II. Thus active RNA polymerase I appears to be required for the formation of the nucleolus as its major component, and DNA topoisomerases appear to be required for the folding of rDNA and RNA polymerase I molecules into the functional organization of nucleolar genes.     

A functional 125-kDa core polypeptide of fission yeast DNA topoisomerase II.

We purified fission yeast DNA topoisomerase II (topo II) to apparent homogeneity. It consists of a single 165-kDa polypeptide in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and, upon treatment with a bifunctional reagent, doubles its molecular weight. Limited proteolysis of intact topo II by papain produces a 125-kDa core, which lacks the N-terminal 75 and the C-terminal approximately 260 amino acids but still contains regions similar to those of bacterial or phage T4 topo II subunits. The core retains relaxing and unknotting activities. Further digestion inactivates the core, cleaving it at the middle of the GyrB-like domain and at the beginning of the GyrA-like domain. Therefore, papain appears to cleave spatially distinct subdomains of topo II. We made top2 mutant genes deleted of the C-terminal 286 or N-terminal 74 amino acids, which can substitute for the wild-type top2+ gene in mitosis and meiosis. However, a mutant containing deletions of both termini cannot rescue the top2 null mutant, despite the fact that the product is enzymatically active. Therefore, the top2 product of the doubly truncated gene may not fulfill all of the in vivo requirements for top2+ function.     

A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III.

RNA polymerase II (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes. The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species. We have initiated a genetic analysis of RNAPII by introducing mutations in RPO21, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae. We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII. We found that increased expression of wild-type RPO26, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo21-4 temperature-sensitive mutation. Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RPO26 gene product. One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo21-4. These results support the idea that the RPO26 and RPO21 gene products interact.     

The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.     

Sequence-specific interaction of sigma subunit of E. coli RNA polymerase with DNA.

The interaction of sigma subunit of E. coli RNA polymerase with DNA, either double or single-stranded, and with two inhibitors of RNA synthesis was investigated by using antibodies directed against the subunit. Free sigma subunit was shown to interact with poly(dA), poly(dT), poly(dAC).poly(dGT), T7 DNA and, to a lesser degree, with lambda DNA. When the sigma subunit forms part of the holo enzyme, sigma also interacts with poly(dG).poly(dC). Rifampicin and streptolydigin interact with sigma in the holo enzyme and with free and core bound sigma subunit, respectively. The results suggest that sigma recognizes mainly AC-GT-sequences in double-stranded DNA. The findings are correlated with the base composition in RNA polymerase binding regions of promoters and suggest at least a general interaction between sigma subunit and single-stranded DNA in open complexes.     

RPA190, the gene coding for the largest subunit of yeast RNA polymerase A

Yeast RNA polymerases are being extensively studied at the gene level. The entire gene encoding the largest subunit of RNA polymerase A, A190, was isolated and characterized in detail. Southern hybridization and gene disruption experiments showed that the RPA190 gene is unique in the haploid yeast genome and essential for cell viability. Nuclease S1 mapping was used to identify mRNA 5' and 3' termini. RPA190 encodes a polypeptide chain of 186,270 daltons in a large uninterrupted reading frame. A dot matrix comparison of the deduced amino acid sequence of subunit A190 with Escherichia coli beta' and cognate subunits B220 and C160 from yeast RNA polymerases B and C showed a conserved pattern of homology regions (I-VI). A potential DNA-binding site (zinc-binding motif) is conserved in the N-terminal region I. Remarkably, the A190 subunit does not harbor the heptapeptide repeated sequence present in the B220 subunit. The sequence of the A190 subunit diverges from B220 and C160 by the presence of two hydrophilic domains inserted between homology regions I and II, and V and VI. From their codon usage and third base pyrimidine bias, RNA polymerase genes RPA190, RPB220, RPC160, and RPC40 fall among yeast genes expressed at an average level. The RPA190 5'-flanking region contains features present in other polymerase genes that might function in regulation.