Zinc stoichiometry of yeast RNA polymerase II and characterization of mutations in the zinc-binding domain of the largest subunit

Atomic absorption spectroscopy demonstrated that highly purified RNA polymerase II from the yeast Saccharomyces cerevisiae binds seven zinc ions. This number agrees with the number of potential zinc-binding sites among the 12 different subunits of the enzyme and with our observation that the ninth largest subunit alone is able to bind two zinc ions. The zinc-binding motif in the largest subunit of the enzyme was investigated using mutagenic analysis. Altering any one of the six conserved residues in the zinc-binding motif conferred either a lethal or conditional phenotype, and zinc blot analysis indicated that mutant forms of the domain had a 2-fold reduction in zinc affinity. Mutations in the zinc-binding domain reduced RNA polymerase II activity in cell-free extracts, even though protein blot analysis indicated that the mutant subunit was present in excess of wild-type levels. Purification of one mutant RNA polymerase revealed a subunit profile that was wild-type like with the exception of two subunits not required for core enzyme activity (Rpb4p and Rpb7p), which were missing. Core activity of the mutant enzyme was reduced 20-fold. We conclude that mutations in the zinc-binding domain can reduce core activity without altering the association of any of the subunits required for this activity.     

Conservation of a DNA-binding site in the largest subunit of eukaryotic RNA polymerase II

Using a monoclonal antibody to a DNA-binding site of calf RNA polymerase II, we found that this site occurs on the largest subunit and is structurally similar in RNA polymerase II of widely divergent eukaryotes. In immuno-blotting of electrophoretically separated subunits, the monoclonal antibody recognized a determinant on the largest polypeptide of all RNA eukaryotic polymerase II forms tested, with a preference for the IIA enzyme subunit of 215 X 10(3) Mr over the partially proteolyzed 180 X 10(3) Mr form. This site is conserved on human, chicken, Drosophila, wheat germ and yeast RNA polymerase II, all of which reacted strongly with the monoclonal antibody. These results contrasted with those obtained with polyclonal antibodies to non-functional determinants of the calf enzyme. The reactivity of the polyclonal antibody with eukaryotic RNA polymerase II steadily decreased with increasing evolutionary distance from the original antigen; the yeast enzyme showed no cross-reactivity. These results suggest that a basic functional feature of eukaryotic RNA polymerase II has been strongly conserved and support the view that divergence of RNA polymerase II has taken place mainly in other, perhaps regulatory, sites of the enzyme.     

An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension.     

Genetic variation in RPOIILS gene encoding RNA polymerase II largest subunit from Leishmania major

Leishmaniasis is a geographically widespread severe disease which includes visceral leishmaniasis, cutaneous leishmaniasis (CL). There are 350 million people at risk in over 80 countries. In the Old World, CL is usually caused by Leishmania major, Leishmania tropica, and Leishmania aethiopica complex which 90 % of cases occurring in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil, and Peru. Recently, some reports showed that some strains of L. major have internal transcribed space (ITS-1) with differential size exhibiting homology with the related gene in a divergent genus of kinetoplastida, the Crithidia. This prompted us to analyze the mentioned gene in 100 isolates obtained from patients with suspected CL. After obtaining samples from 100 patients, DNA extraction was performed and ITS-1 was analyzed using PCR–RFLP. These samples were sequenced for verifying their homology. Then, RPOIILS gene was analyzed in the samples that their ITS-1 gene exhibiting homology with the related gene in Crithidia. Results showed that 10 % of the isolates have ITS-1 exhibiting different size with the routine ones. Sequencing of them showed their similarity to the one from Crithidia fasciculata. RPOIILS gene encoding RNA polymerase II largest subunit analysis showed genetic diversity. This study might also help in solving the problems concerning Leishmaniasis outbreak currently facing in Iran and some other endemic regions of the world.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila

Summary We have characterized RpII215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster. DNA sequencing and nuclease S1 analyses provided the primary structure of this gene, its 7 kb RNA and 215 kDa protein products. The amino-terminal 80% of the subunit harbors regions with strong homology to the subunit of Escherichia coli RNA polymerase and to the largest subunits of other eukaryotic RNA polymerases. The carboxyl-terminal 20% of the subunit is composed of multiple repeats of a seven amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The homology domains, as well as the unique carboxyl-terminal structure, are considered in the light of current knowledge of RNA polymerase II and the properties of its largest subunit. Additionally, germline transformation demonstrated that a 9.4 kb genomic DNA segment containing the -amanitinresistant allele, RpII215 ^C4 , includes all sequences required to produce amanitin-resistant transformants.     

Size heterogeneity of the largest subunit of nuclear RNA polymerase II. An immunological analysis

Antibodies raised against the 180-kDa subunit of cauliflower RNA polymerase II bind selectively to the largest subunit of RNA polymerase II purified from a variety of plant species. The selective binding of this antibody to the largest RNA polymerase II subunit has allowed us to probe for the size of this subunit in crude cell extracts, in fractions containing partially purified RNA polymerase II, and in isolated nuclei. Fractions containing RNA polymerase II were subjected to electrophoresis in the presence of sodium dodecyl sulfate, blotted onto nitrocellulose, and blots were probed with antibody. Immunoglobulin complexes were revealed with 125I-Protein A. Published purification procedures result in rapid conversion of a 220-kDa subunit to a 180-kDa polypeptide, but purification at high pH (pH 9.0) retards this proteolysis. RNA polymerase II associated with isolated nuclei is largely protected from proteolytic degradation, and a 240-kDa polypeptide as well as a 220-kDa polypeptide can be detected. These results suggest that the 180-kDa subunit of RNA polymerase II arises artificially during cell lysis and enzyme purification, and that even the 220-kDa polypeptide may be a degradation product of a 240-kDa polypeptide in plants.