Evolution of RNA polymerases and branching patterns of the three major groups of archaebacteria

Summary The amino acid sequences of the largest subunits of the RNA polymerases I, II, and III from eukaryotes were compared with those of archaebacterial and eubacterial homologs, and their evolutionary relationships were analyzed in detail by a recently developed tree-making method, the likelihood method of protein phylogeny, as well as by the neighbor-joining method and the parsimony method, together with bootstrap analyses. It was shown that the best tree topologies predicted by the first two methods are identical, whereas the last one predicts a distinct tree. The maximum likelihood tree revealed that, after the separation from archaebacteria, the three eukaryotic RNA polymerases diverged from an ancestral precursor in the eukaryotic lineage. This result is contrasted with the published result showing multiple origins for the three eukaryotic polymerases. It was shown that eukaryotic RNA polymerase I evolved much more rapidly than RNA polymerases II and III: The N-terminal half of RNA polymerase I shows an extraordinarily high evolutionary rate, possibly due to relaxed functional constraints. In contrast the evolutionary rate of archaebacterial RNA polymerase is remarkably limited. In addition, including the second largest subunit of the RNA polymerase, a detailed analysis for the branching pattern of the three major groups of archaebacteria was carried out by the maximum likelihood method. It was shown that the three major groups of archaebacteria are likely to form a single cluster; that is, archaebacteria are likely to be monophyletic as originally proposed by Woese and his colleagues.     

The phylogenetic relations of DNA-dependent RNA polymerases of archaebacteria, eukaryotes, and eubacteria

Unrooted phylogenetic dendrograms were calculated by two independent methods, parsimony and distance matrix analysis, from an alignment of the derived amino acid sequences of the A and C subunits of the DNA-dependent RNA polymerases of the archaebacteria Sulfolobus acidocaldarius and Halobacterium halobium with 12 corresponding sequences including a further set of archaebacterial A+C subunits, eukaryotic nuclear RNA polymerases, pol I, pol II, and pol III, eubacterial beta' and chloroplast beta' and beta" subunits. They show the archaebacteria as a coherent group in close neighborhood of and sharing a bifurcation with eukaryotic pol II and (or) pol IIIA components. The most probable trees show pol IA branching off from the tree separately at a bifurcation with the eubacterial beta' lineage. The implications of these results, especially for understanding the possibly chimeric origin of the eukaryotic nuclear genome, are discussed.     

Archaebacteria and eukaryotes possess DNA-dependent RNA polymerases of a common type

DNA-dependent RNA polymerases of archaebacteria not only resemble the nuclear RNA polymerases of eukaryotes rather than the eubacterial enzymes in their complex component patterns but also show striking immunochemical, i.e., structural, homology with the eukaryotic polymerases at the level of single components. Thus, eukaryotic and archaebacterial RNA polymerases are indeed of the same type, distinct from the eubacterial enzymes, which, however, are also derived from a common ancestral structure.     

Antigenic homology of eukaryotic RNA polymerases

Facilitated by an improved enzyme purification procedure, antisera to calf thymus DNA-dependent RNA polymerase II was prepared in hens. Using immunoprecipitation and inhibition of enzymatic activity the immunological properties of several eukaryotic RNA polymerases were examined. Purified calf thymus and rat liver polymerase II exhibited antigenic homology. The partially purified amphibian ($\text{Xenopus laevis}$) and protozoan ($\text{Tetrahymena pyriformis}$) polymerase II had reduced crossreactivities. Calf thymus polymerase I also shared antigenic homology with the form II enzymes.     

α-Amanitin-sensitive DNA-dependent RNA polymerase I from cherry salmon (Oncorhynchus masou) liver nuclei

DNA-dependent RNA polymerases I and II were purified approximately 3900- and 13300 fold, respectively, from a sonicated nuclear extract of the cherry salmon liver by column chromatographies on DEAE-Sephadex, heparin-Sepharose and DNA-cellulose. The RNA polymerases were examined with respect to template-specificity, the effects of Mn²⁺, Mg²⁺ and ammonium sulfate, α-amanitin sensitivity. Results showed that the RNA polymerase I differed from other eukaryotic RNA polymerase I in α-amanitin sensitivity.     

Partial purification and characterization of DNA-dependent RNA polymerases I and II from cherry salmon (Oncorhynchus masou)

1. DNA-dependent RNA polymerases I and II were purified approx 3900- and 13,000-fold, respectively, from sonicated nuclear extract of cherry salmon (Oncorhynchus masou) liver by DEAE-Sephadex, heparin-Sepharose and DNA-cellulose column chromatography. 2. The purified RNA polymerases exhibited a requirement for four kinds of ribonucleoside 5'-triphosphates, an exogeneous template and divalent cation. 3. The activities of RNA polymerases I and II were inhibited by Actinomycin D (24 micrograms/ml) but not by Rifampicin (200 micrograms/ml). 4. RNA polymerase I preferred native DNA as template, while polymerase II preferred single-stranded DNA. 5. RNA polymerase II was inhibited by a low concentration of alpha-amanitin (0.02 micrograms/ml). RNA polymerase I was also inhibited by the relatively high concentration of alpha-amanitin (IC50 = 100 micrograms/ml and IC70 = 750 micrograms/ml). 6. RNA polymerases from cherry salmon exhibited a higher activity at low temperature than from rat liver.     

An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.

Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.     

Immunological relationships between Artemia RNA polymerases and between RNA polymerases II from different eukaryotic organisms

Summary Rabbit antibodies against Artemia RNA polymerase II have been raised and utilized to study the immunological relationships between the subunits from RNA polymerases I, II and III from this organism and RNA polymerase II from other eukaryotes. We describe here for the first time the subunit structure of Artemia RNA polymerases I and III. These enzymes have 9 and 13 subunits respectively. The anti-RNA polymerase II antibodies recognize two subunits of 19.4 and 18 kDa common to the three enzymes, and another subunit of 25.6 kDa common to RNA polymerases II and III. The antibodies against Artemia RNA polymerase II also react with the subunits of high molecular weight and with subunits of around 25 and 33 kDa of RNA polymerase II from other eukaryotes (Drosophila melanogaster, Chironomus thummi, triticum (wheat) and Rattus (rat)). This interspecies relatedness is a common feature of eukaryotic RNA polymerases.Abbreviations RNAp RNA polymerase - DPT diazophenylthioether - SDS sodium dodecylsulfate     

Differential inhibition of mammalian ribonucleic acid polymerases by an exotoxin from Bacillus thuringiensis. The direct observation of nucleoplasmic ribonucleic acid polymerase activity in intact nuclei

The effects of the exotoxin from Bacillus thuringiensis on DNA-dependent RNA polymerases from rat liver were examined. The exotoxin inhibits all RNA polymerase activity at both low and high ionic strength in intact nuclei, and soluble enzymes are similarly affected. This inhibition is relieved by ATP. Dephosphorylated exotoxin did not inhibit the soluble enzymes. Nucleolar and nucleoplasmic RNA polymerases respond to different concentration ranges of exotoxin, and the compound can be used in intact nuclei to isolate the nucleoplasmic activity.     

The RNA Polymerase Structure–Function Study (1962–2001)

The study of RNA polymerase initiated by R.B. Khesin has been conducted for about forty years at the laboratory founded by him (since 1989, in collaboration with A. Goldfarb's laboratory). Genetic methods are used in combination with methods of the specific chemical crosslinks of nucleic acids with proteins. The paper assesses the main results of the study in comparison with the recent high-resolution X-ray crystallographic data. A short comparative summary of the RNA polymerase structure is presented for bacteria, archaebacteria, and eukaryotic organelles and nuclei. A brief history of the RNA polymerase study is also given.     

The RNA polymerase structure-activity studies (1962-2001)

The study of RNA polymerase initiated by R.B. Khesin is conducted for about forty years at the laboratory founded by him (since 1989, in collaboration with A. Goldfarb's laboratory). Genetic methods are used in combination with methods of the specific chemical crosslinks of nucleic acids with proteins. The paper assesses the main results of the study in comparison with the X-ray crystallographic data of high resolution obtained recently. A short comparative summary of the RNA polymerase structure has been done for bacteria, archaebacteria, and eukaryotic organelles and nuclei. A brief history of the RNA polymerase study is also presented.     

DNA polymerase alpha from normal rat liver is different than DNA polymerases alpha from Morris hepatoma strains

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.