Genes for DNA-dependent RNA polymerase components B, A, and C from the archaebacterium Sulfolobus acidocaldarius and for components B", B', A, and C from the archaebacterium Halobacterium halobium were cloned and sequenced. They are organized in gene clusters in the order above, which corresponds to the order of the homologous rpoB and IpoC genes in the corresponding operon of the Escherichia coli genome. Derived amino acid sequences of archaebacterial components A and C were aligned with each other and with the sequences of corresponding (largest) subunits from the archaebacterium Methanobacterium thermoautotrophicum, with sequences of various eukaryotic nuclear RNA polymerases I, 1, and Ill, and with the sequence ofthe 1' component from E. coli Phylogenetic relations between the primary kingdoms of life have been established using rRNA as an evolutionary chronometer (1, 2). RNA polymerase appears to be an appropriate alternative because it is ubiquitous, highly conserved, and a complex macromolecule. We have cloned and sequenced the genes for the large RNA polymerase components of two phylogenetically distant archaebacteria: the sulfur-dependent extreme thermophile Sulfolobus acidocaldarius (Su. acidocaldarius) and the extreme halophile Halobacterium halobium. Eukaryotes possess three specialized nuclear RNA polymerases (pol I, II, and III) thought to be derived from one ancestral "urkaryotic" enzyme by diversification (3). Archaebacteria should provide an independent outside reference, in addition to eubacteria, for elucidating the origin of the eukaryotic lineage.DNA-dependent RNA polymerases from archaebacteria are of two types (4, 5). The BAC type (largest subunits arranged in order of decreasing size) is found in the sulfurdependent extreme thermophiles (6), including the Thermococcales (7), and in the genus Thermoplasma, which belongs to the second major branch of the archaebacterial kingdom (1). The AB'B"C type, characterized by a split B component, is common to the methanogens and extreme halophiles, other representatives of the second major branch. Immunochemical cross-reactivity indicates that the two (or three) largest components of eubacterial, eukaryotic nuclear, and archaebacterial RNA polymerases are homologous (4, 8). Interkingdom homologies between smaller components, including the archaebacterial C subunits, have not yet been ascertained.The complex component patterns of archaebacterial RNA polymerases, comprising around 10 subunits each, resemble those of the nuclear eukaryotic rather than the more streamlined eubacterial enzymes. Accordingly, immunochemical cross-reactivity was larger between archaebacterial and eukaryotic components than between archaebacterial and eubacterial components (8), and the consensus sequences of putative promoters of archaebacterial stable RNA and protein genes (9, 10) are strikingly similar to those of eukaryotic promoters for RNA polymerase II (11).Here we compare the derived amino acid sequences of components A and C from three archaebacteria with eac...

The proteasome or multicatalytic proteinase is a high molecular mass multisubunit complex ubiquitous in eukaryotes but also found in the archaebacterial proteasome is made of two different subunits only, and yet the complexes are almost identical in size and shape. Cloning and sequencing the gene encoding the small (beta) subunit of the T. acidophilum complex completes the primary structure of the archaebacterial proteasome. The similarity of the derived amino acid sequences of 233 (alpha) and 211 (beta) residues, respectively, indicates that they arose from a common ancestral gene. All the sequences of proteasomal subunits from eukaryotes available to date can be related to either the alpha-subunit or beta-subunit of the T. acidophilum "Urproteasome", and they can be distinguished by means of a highly conserved N-terminal extension, which is characteristic for alpha-type subunits. On the basis of circumstantial evidence we suggest that the alpha-subunits have regulatory and targeting functions, while the beta-subunits carry the active sites.

The proteasome or multicatalytic proteinase from the archaebacterium Thermoplasma acidophilum is a 700 kDa multisubunit protein complex. Unlike proteasomes from eukaryotic cells which are composed of 10–20 different subunits, the Thermoplasma proteasome is made of only two types of subunit, alpha and beta, which have molecular weights of 25.8 and 22.3 kDa, respectively. In this communication we present a three‐dimensional stoichiometric model of the archaebacterial proteasome deduced from electron microscopic investigations. The techniques which we have used include image analysis of negatively stained single particles, image analysis of metal decorated small three‐dimensional crystals after freeze‐etching and STEM mass measurements of freeze‐dried particles. The archaebacterial and eukaryotic proteasomes are almost identical in size and shape; the subunits are arranged in four rings which are stacked together such that they collectively form a barrel‐shaped complex. According to a previous immunoelectron microscopic investigation, the alpha‐subunits form the two outer rings of the stack, while the two rings composed of beta‐subunits, which are supposed to carry the active sites, are sandwiched between them. Each of the alpha‐ and beta‐rings contains seven subunits; hence the stoichiometry of the whole proteasome is alpha 14 beta 14 and the symmetry is 7‐fold. Image simulation experiments indicate that the alpha‐ and beta‐subunits are not in register along the cylinder axis; rather it appears that the beta‐rings are rotated with respect to the alpha‐rings by approximately 25 degrees. In contrast to some previous reports we have not been able to find stoichiometric amounts of RNA associated with highly purified proteolytically active proteasome preparations.

Gene organization, gene structure, especially regarding transcription and translation signals, and the structure of essential components of the gene expression machinery of archaebacteria are compared with those of eubacteria and eukaryotes. Many features of the genetic machinery of archaebacteria are shared either with eubacteria or with eukaryotes. For example, the translation signals including ribosome-binding sites are the same as in eubacteria, but the consensus sequence of archaebacterial promoters closely resembles that of the eukaryotic polymerase I1 promoters.Archaebacterial genes can be organized in transcription units resembling those of eubacteria. But the sequences of several protein components of the genetic machinery have strikingly more homology with those of their eukaryotic than with those of their eubacterial correspondents.The sequences of the large components of DNA-dependent RNA polymerases of archaebacteria closely resemble those of the eukaryotic RNA polymerases I1 and, somewhat less, 111. In a dendrogram calculated from percentage homology data, the eukaryotic RNA polymerase I component A shares a branching point with the eubacterial component. The implications of these findings for the origin and the evolution of the eukryotic ancestry are discussed.The comparison of sequences of rRNAs 11-61, tRNAs [7, 81, G. Piihler & F. Gropp, unpublished) and a characteristic pattern of few unique and many quasi-eubacterial or quasieukaryotic feature designs (see e.g. [l, 2, 121) indicates that archaebacteria form a monophyletic holophyletic (singlerooted, comprising all descendants) rather than a paraphyletic (not comprising all descendants) group of organisms. The possibility that eubacteria and eukaryotes might have arisen from within the archaebacteria is not entirely excluded but has become remote, because each of the unrooted phylogenetic trees derived from these data shows a significant though short distance between the lowest branching point within the archaebacteria and the trifurcation point between the three kingdoms [2, unpublished). Thus at present the major issue of comparative molecular biology is to establish the branching order in the early evolution of organisms, i.e. to determine the roots of the phylogenetic trees, a task, which in the absence of paleontological evidence appears at least difficult.The comparative molecular biology of archaebacteria has made its most remarkable progress in the fields of molecular phylogeny and molecular genetics (see [13 -171). Recent developments in our understanding of structure and function, especially expression of archaebacterial genes and genomes, and the nature of the components of the genetic machinery call for comparative evaluation. This overview discusses these new findings with an emphasis on areas in which our laboratory is directly involved.

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