Prokaryotic and eukaryotic RNA polymerases have homologous core subunits.

Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases.     

Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer.

The active conformation of native peroxisomal 3-ketoacyl-CoA thiolases (EC 2.3.1.16) is homodimeric. We have previously shown that a truncated Saccharomyces cerevisiae thiolase lacking its first 16 N-terminal amino acids fails to be translocated into peroxisomes but assembles into an enzymatically active form in the cytoplasm of a strain with a disrupted nuclear thiolase gene. We now report that when truncated thiolase is cosynthesized with full-length thiolase, approximately 50% of truncated thiolase cofractionates with the full-length thiolase to fractions enriched for peroxisomes and is translocated into peroxisomes as shown by its protection from the action of external proteases. We constructed an immunologically distinct cytosolic variant of thiolase by adding an influenza hemagglutinin epitope tag to the N terminus of the truncated thiolase. In a strain simultaneously expressing the full-length, truncated, and epitope-tagged truncated thiolases, we demonstrated that normally untargeted thiolase subunits are efficiently translocated into peroxisomes by dimerization with full-length thiolase subunits. Even though truncated and epitope-tagged truncated thiolase subunits are translocated into peroxisomes in this strain, only the full-length thiolase subunit can be coimmunoprecipitated with the epitope-tagged truncated thiolase subunit from the peroxisomal matrix. This observation suggests that interactions between thiolase subunits are not disrupted during translocation.     

RNase-like domain in DNA-directed RNA polymerase II.

DNA-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae. Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved. This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity. The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I. The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function.     

Analysis of Schizosaccharomyces pombe mediator reveals a set of essential subunits conserved between yeast and metazoan cells

With the identification of eight new polypeptides, we here complete the subunit characterization of the Schizosaccharomyces pombe RNA polymerase II holoenzyme. The complex contains homologs to all 10 essential gene products present in the Saccharomyces cerevisiae Mediator, but lacks clear homologs to any of the 10 S. cerevisiae components encoded by nonessential genes. S. pombe Mediator instead contains three unique components (Pmc2, -3, and -6), which lack homologs in other cell types. Presently, pmc2(+) and pmc3(+) have been shown to be nonessential genes. The data suggest that S. pombe and S. cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10 essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated.     

A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II.

Purified eukaryotic nuclear RNA polymerase II consists of three subspecies that differ in the apparent molecular masses of their largest subunit, designated IIo, IIa, and IIb for polymerase species IIO, IIA, and IIB, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. We present here the amino acid composition of calf thymus subunits IIa and IIb and the C-terminal amino acid sequence of subunit IIa (IIo) inferred from the nucleotide sequence of part of the mouse gene encoding this RNA polymerase subunit. The calculated amino acid composition of the peptide unique to subunit IIa indicates that subunit IIa contains a domain rich in serine, proline, threonine, and tyrosine. The sequence at the 3' end of the mouse RNA polymerase II largest subunit gene reveals that the C-terminal domain consists of 52 repeats of a seven amino acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This sequence is also unusual in that it contains a high percentage of potential phosphorylation sites.     

Interactions among the protein and RNA subunits of Saccharomyces cerevisiae nuclear RNase P

Ribonuclease P (RNase P) is a ubiquitous endoribonuclease that cleaves precursor tRNAs to generate mature 5' termini. Although RNase P from all kingdoms of life have been found to have essential RNA subunits, the number and size of the protein subunits ranges from one small protein in bacteria to at least nine proteins of up to 100 kDa. In Saccharomyces cerevisiae nuclear RNase P, the enzyme is composed of ten subunits: a single RNA and nine essential proteins. The spatial organization of these components within the enzyme is not yet understood. In this study we examine the likely binary protein-protein and protein-RNA subunit interactions by using directed two- and three-hybrid tests in yeast. Only two protein subunits, Pop1p and Pop4p, specifically bind the RNA subunit. Pop4p also interacted with seven of the other eight protein subunits. The remaining protein subunits all showed one or more specific protein-protein interactions with the other integral protein subunits. Of particular interest was the behavior of Rpr2p, the only protein subunit found in RNase P but not in the closely related enzyme, RNase MRP. Rpr2p interacts strongly with itself as well as with Pop4p. Similar interactions with self and Pop4p were also detected for Snm1p, the only unique protein subunit so far identified in RNase MRP. This observation is consistent with Snm1p and Rpr2p serving analogous functions in the two enzymes. This study provides a low-resolution map of the multisubunit architecture of the ribonucleoprotein enzyme, nuclear RNase P from S. cerevisiae.     

Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits

We have determined the nucleotide sequence of a region of the vaccinia virus genome encoding RNA polymerase subunits of 22 and 147 kDa and have mapped the 5' and 3' ends of the two mRNAs. The predicted amino acid sequence of the vaccinia 147-kDa subunit shows extensive homology with the largest subunit of Escherichia coli RNA polymerase, yeast RNA polymerases II and III, and Drosophila RNA polymerase II. The regions of homology between the five RNA polymerases are subdivided into five separate domains that span most of the length of each. A sixth domain shared by the vaccinia and the eukaryotic polymerases is absent from the E. coli sequence. In all specified regions, the vaccinia large subunit has greater homology with eukaryotic RNA polymerases II and III than with the E. coli polymerase. Vaccinia virus and eukaryotic RNA polymerases may therefore have evolved from a common ancestral gene after the latter diverged from prokaryotes.     

Molecular cloning and expression of the Saccharomyces cerevisiae RFC3 gene, an essential component of replication factor C.

Yeast replication factor C (RF-C) is a multi-polypeptide complex required for processive DNA replication by DNA polymerases delta and epsilon. The gene encoding the 40-kDa subunit of the Saccharomyces cerevisiae RF-C (RFC3) has been cloned. The RFC3 gene is required for yeast cell growth and has been mapped to the left arm of chromosome XIV. The deduced amino acid sequence of the RFC3 gene shows a high homology to the 36-, 37-, and 40-kDa subunits of human RF-C (also called activator 1), with the highest homology to the 36-kDa subunit. Among the conserved regions are the A motif of ATP binding proteins; the "DEAD box," common to DNA helicases and other ATPases; and the "RFC box," an approximately 15-amino acid domain virtually identical in the yeast and human RF-C subunits. Limited homology to the functional homologs of the Escherichia coli replication apparatus was also observed. The steady-state mRNA levels of RFC3 do not change significantly during the mitotic cell cycle of yeast. The intact form of the RFC3 gene product (Rfc3p) has been overproduced in E. coli and purified to homogeneity. Purified Rfc3p has an ATPase activity that is markedly stimulated by single-stranded DNA but not by double-stranded DNA or RNA.     

A putative ATP-dependent RNA helicase involved in Saccharomyces cerevisiae ribosome assembly.

We have isolated a cold-sensitive mutant of Saccharomyces cerevisiae in which there is a deficit of 60S ribosomal subunits. Cold sensitivity and the assembly defect are recessive and cosegregate, defining a single essential gene that we designated DRS1 (deficiency of ribosomal subunits). The wild-type DRS1 gene was cloned by complementation of the cold-sensitive phenotype of drs1. Sequence analysis reveals a high degree of similarity to a family of proteins that are thought to function as ATP-dependent RNA helicases. Pulse-chase analysis of ribosomal RNA synthesis and processing indicates that the drs1 mutant accumulates the 27S precursor of the mature 25S rRNA. These results suggest that, as in pre-mRNA splicing, RNA helicase activities are involved in ribosomal RNA processing.