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.     

CCA-adding enzymes and poly(A) polymerases are all members of the same nucleotidyltransferase superfamily: characterization of the CCA-adding enzyme from the archaeal hyperthermophile Sulfolobus shibatae.

We describe the purification, cloning, and characterization of the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyl transferase] from the thermophilic archaebacterium, Sulfolobus shibatae. Characterization of an archaeal CCA-adding enzyme provides formal proof that the CCA-adding activity is present in all three contemporary kingdoms. Antibodies raised against recombinant, expressed Sulfolobus CCA-adding enzyme reacted specifically with the 48-kDa protein and fully depleted all CCA-adding activity from S. shibatae crude extract. Thus, the cloned cca gene encodes the only CCA-adding activity in S. shibatae. Remarkably, the archaeal CCA-adding enzyme exhibits no strong homology to either the eubacterial or eukaryotic CCA-adding enzymes. Nonetheless, it does possess the active site signature G[SG][LIVMFY]xR[GQ]x5,6D[LIVM][CLIVMFY]3-5 of the nucleotidyltransferase superfamily identified by Holm and Sander (1995, Trends Biochem Sci 20:345-347) and sequence comparisons show that all known CCA-adding enzymes and poly(A) polymerases are contained within this superfamily. Moreover, we propose that the superfamily can now be divided into two (and possibly three) subfamilies: class I, which contains the archaeal CCA-adding enzyme, eukaryotic poly(A) polymerases, and DNA polymerase beta; class II, which contains eubacterial and eukaryotic CCA-adding enzymes, and eubacterial poly(A) polymerases; and possibly a third class containing eubacterial polynucleotide phosphorylases. One implication of these data is that there may have been intraconversion of CCA-adding and poly(A) polymerase activities early in evolution.     

Sequence motifs that distinguish ATP(CTP):tRNA nucleotidyl transferases from eubacterial poly(A) polymerases

ATP(CTP):tRNA nucleotidyl transferases, tRNA maturing enzymes found in all organisms, and eubacterial poly(A) polymerases, enzymes involved in mRNA degradation, are so similar that until now their biochemical functions could not be distinguished by their amino acid sequence. BLAST searches and analysis with the program "Sequence Space" for the prediction of functional residues revealed sequence motifs which define these two protein families. One of the poly(A) polymerase defining motifs specifies a structure that we propose to function in binding the 3' terminus of the RNA substrate. Similar motifs are found in other homopolyribonucleotidyl transferases. Phylogenetic classification of nucleotidyl tranferases from sequenced genomes reveals that eubacterial poly(A) polymerases have evolved relatively recently and are found only in a small group of bacteria and surprisingly also in plants, where they may function in organelles.     

Mechanism and evolution of DNA primases

DNA primase synthesizes short RNA primers that replicative polymerases further elongate in order to initiate the synthesis of all new DNA strands. Thus, primase owes its existence to the inability of DNA polymerases to initiate DNA synthesis starting with 2 dNTPs. Here, we discuss the evolutionary relationships between the different families of primases (viral, eubacterial, archael, and eukaryotic) and the catalytic mechanisms of these enzymes. This includes how they choose an initiation site, elongate the growing primer, and then only synthesize primers of defined length via an inherent ability to count. Finally, the low fidelity of primases along with the development of primase inhibitors is described.     

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.     

Studies on DNA polymerases and topoisomerases in archaebacteria

We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.     

Sequence comparison of glyceraldehyde-3-phosphate dehydrogenases from the three urkingdoms: evolutionary implication

The primary structure of the glyceraldehyde-3-phosphate dehydrogenase from the archaebacteria shows striking deviation from the known sequences of eubacterial and eukaryotic sequences, despite unequivocal homologies in functionally important regions. Thus, the structural similarity between the eubacterial and eukaryotic enzymes is significantly higher than that between the archaebacterial enzymes and the eubacterial and eukaryotic enzymes. This preferred similarity of eubacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenase structures does not correspond to the phylogenetic distances among the three urkingdoms as deduced from comparisons of ribosomal ribonucleic acid sequences. Indications will be presented that the closer relationship of the eubacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenase resulted from a gene transfer from eubacteria to eukaryotes after the segregation of the three urkingdoms.     

Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the fungus Podospora anserina

Three DNA-dependent RNA polymerases have been isolated and partially purified from the mycelium of the fungus Podospora anserina. Separated by DEAE-Sephadex chromatography, they have been designated RNA polymerases I, II, and III according to their order of elution. Their catalytic properties and alpha-amanitin sensitivity are in agreement with those of the homologous enzymes found in other eukaryotic organisms. The three enzymes exhibit rather sharp monophasic ammonium sulfate dependence with optima which are, respectively, 0.035 M, 0.050 M, and 0.075 M. Enzyme I has the largest Mn2+/Mg2+ activity ratio, shows a marked preference for native DNA, and is insensitive to alpha-amanitin. Enzyme III uses poly(dA-dT) in preference to native DNA as template and is only partially sensitive to alpha-amanitin. Enzyme II is sensitive to alpha-amanitin, but high concentrations of the toxin are required for inhibition compared to other eukaryotic class II enzymes. Three similar RNA polymerases with comparable levels of activity were found in the temperature-dependent VR strain when cellular incompatibility, leading to a rapid cessation of RNA synthesis, was induced.     

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     

Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase III from the mouse plasmacytoma, MOPC 315.

Class III DNA-dependent RNA polymerases were purified from the mouse plasmacytoma, MOPC 315. RNA polymerases IIIA and IIIB were solubilized from a whole cell extract and resolved by chromatography on DEAE-Sephadex. Chromatography on DEAE-cellulose, DEAE-Sephadex, CM-Sephadex, and phosphocellulose ion exchange resins and sedimentation in sucrose density gradients yielded chromatographically homogeneous Enzymes IIIA and IIIB which were purified approximately 22,000 and 53,000-fold respectively, relative to whole cell extracts. The specific activity of these enzymes was comparable to that reported for other purified eukaryotic RNA polymerases. Sucrose gradient sedimentation analysis suggested a molecular weight of approximately 650,000 for each of the class III enzymes.