High-resolution structure of an archaeal zinc ribbon defines a general architectural motif in eukaryotic RNA polymerases.

BACKGROUND: Transcriptional initiation and elongation provide control points in gene expression. Eukaryotic RNA polymerase II subunit 9 (RPB9) regulates start-site selection and elongational arrest. RPB9 contains Cys4 Zn(2+)-binding motifs which are conserved in archaea and homologous to those of the general transcription factors TFIIB and TFIIS. RESULTS: The structure of an RPB9 domain from the hyperthermophilic archaeon Thermococcus celer was determined at high resolution by NMR spectroscopy. The structure consists of an apical tetrahedral Zn(2+)-binding site, central beta sheet and disordered loop. Although the structure lacks a globular hydrophobic core, the two surfaces of the beta sheet each contain well ordered aromatic rings engaged in serial edge-to-face interactions. Basic sidechains are clustered near the Zn(2+)-binding site. The disordered loop contains sidechains conserved in TFIIS, including acidic residues essential for the stimulation of transcriptional elongation. CONCLUSIONS: The planar architecture of the RPB9 zinc ribbon-distinct from that of a conventional globular domain-can accommodate significant differences in the alignment of polar, non-polar and charged sidechains. Such divergence is associated with local and non-local changes in structure. The RPB9 structure is distinguished by a fourth beta strand (extending the central beta sheet) in a well ordered N-terminal segment and also differs from TFIIS (but not TFIIB) in the orientation of its apical Zn(2+)-binding site. Cys4 Zn(2+)-binding sites with distinct patterns of polar, non-polar and charged residues are conserved among unrelated RNAP subunits and predicted to form variant zinc ribbons.

The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

The sequence, and its evolutionary implications, of a Thermococcus celer protein associated with transcription.

Through random search, a gene from Thermococcus celer has been identified and sequenced that appears to encode a transcription-associated protein (110 amino acid residues). The sequence has clear homology to approximately the last half of an open reading frame reported previously for Sulfolobus acidocaldarius [Langer, D. & Zillig, W. (1993) Nucleic Acids Res. 21, 2251]. The protein translations of these two archaeal genes in turn are homologs of a small subunit found in eukaryotic RNA polymerase I (A12.2) and the counterpart of this from RNA polymerase II (B12.6). Homology is also seen with the eukaryotic transcription factor TFIIS, but it involves only the terminal 45 amino acids of the archaeal proteins. Evolutionary implications of these homologies are discussed.

Structure of the archaebacterial 7S RNA molecule.

The genes encoding the 7S RNAs of the archaebacteria Archaeoglobus fulgidus, Methanosarcina acetivorans, Sulfolobus, solfataricus, and Thermococcus celer have been isolated. All four genes occur as single genomic copies and are flanked by sequences containing potential signals for transcriptional promotion and termination. The genes encode RNA molecules approximately 300 nucleotides in length which conform strictly to a model of secondary structure common to all described archaebacterial 7S RNAs. Archaebacterial 7S RNAs exhibit a strong similarity to eukaryotic 7S RNAs in terms of overall secondary structure, while primary sequence conservation is limited to a specific structural domain of the molecule. This domain displays strong primary and secondary structural similarity to features of small eubacterial RNAs, including the small cytoplasmic (sc) RNA of Bacillus subtilis and the 4.5S RNA of Escherichia coli. Conservation of this structural domain among divergent RNA molecules across three kingdoms suggests that these RNAs are the descendants of a unique subcellular structure present before the divergence of the archaebacterial, eubacterial and eukaryotic kingdoms.

Isolation and characterization of the 7S RNA gene from Methanococcus voltae.

The gene encoding the 7S RNA of the archaebacterium Methanococcus voltae has been isolated. The gene occurs as a single copy within the genome and encodes an RNA molecule approximately 300 nucleotides in length. The M. voltae RNA molecule exhibits a strong similarity to both archaebacterial and eucaryotic 7S RNAs in terms of overall secondary structure, while the primary sequence is conserved to a lesser degree. All 7S RNA molecules possess a specific structural domain which is highly conserved in terms of both primary sequence and secondary structure, possibly representing a functional site of the molecule. Conservation of the 7S RNA molecule suggests that it is the descendant of a subcellular structure present before the divergence of the archaebacterial and eucaryotic kingdoms. The M. voltae 7S RNA gene is flanked both 5' and 3' by regions of extremely A + T-rich DNA. The 5'-flanking region contains several potential promoter sequences for archaebacterial RNA polymerases. One such sequence occurs as three direct repeats and bears a strong similarity to sequences found upstream of other archaebacterial genes. The 3'-flanking region contains a strong signal for the termination of transcription.

Sequence of the 16S rRNA gene from the thermoacidophilic archaebacterium Sulfolobus solfataricus and its evolutionary implications.

The sequence of the small-subunit rRNA from the thermoacidophilic archaebacterium Sulfolobus solfataricus has been determined and compared with its counterparts from halophilic and methanogenic archaebacteria, eukaryotes, and eubacteria. The S. solfataricus sequence is specifically related to those of the other archaebacteria, to the exclusion of the eukaryotic and eubacterial sequences, when examined either by evolutionary distance matrix analyses or by the criterion of minimum change (maximum parsimony). The archaebacterial 16S rRNA sequences all conform to a common secondary structure, with the S. solfataricus structure containing a higher proportion of canonical base pairs and fewer helical irregularities than the rRNAs from the mesophilic archaebacteria. S. solfataricus is unusual in that its 16S rRNA-23S rRNA intergenic spacer lacks a tRNA gene.

Putative introns in tRNA genes of prokaryotes.

Sequences of two putative tRNA genes, for serine and leucine, from the archaebacterium Sulfolobus solfataricus contain intervening sequences in the anticodon region. Furthermore, the genes lack encoded CCA 3' termini and are flanked by A + T-rich DNA segments. The introns can both form the same secondary structure, which is a double-helical extension of the anticodon stalk. The resulting structure contains two symmetrically placed 3-base bulge loops, in which are located cleavage sites for the introns. In the one case tested, the gene occurs as a single copy in the genome.

The terminal organization of macronuclear DNA in Oxytricha fallax.

The macronucleus of the hypotrichous ciliate Oxytricha fallax is transcriptionally active and contains linear achromosomal DNA molecules that function as single-gene units. The terminal organization of macronuclear DNA was analyzed by chemical sequencing and S1 mapping. The terminal sequence of total macronuclear DNA was determined for molecules labeled at the 5' or 3' ends. Results indicate that the 5' sequence C4A4C4A4C4 and the 3' sequence G4T4G4T4G4T4G4T4G4 occur at both ends of all DNA molecules in the macronucleus. The discrepancy in the length of the common terminal sequence between the 5' and 3' ends was clarified by a limited S1 digestion experiment, which indicated the existence of a 16 nucleotide long single-stranded tail at the 3' ends.

Putative actin genes in the macronucleus of Oxytricha fallax.

Previous work has shown that the macronuclear DNA of the hypotrichous ciliate Oxytricha fallax is arranged as short achromosomal pieces, 22 to 0.5 kilobase pairs (kb) in length. Micronuclear DNA has a typical chromosomal organization. Macronuclear DNA is derived from micronuclear DNA through a process of polytene chromosome fragmentation with a resultant decrease in DNA sequence complexity. Three putative actin genes have been identified in macronuclear DNA by using a cloned yeast actin gene as a hybridization probe. A restriction fragment of the yeast gene containing both actin coding and noncoding DNA hybridizes strongly to two macronuclear DNA pieces, 1.6 and 1.4 kb in length, and weakly to a 1.2-kb piece. The entire 1.6-kb piece has been cloned in plasmid pBR322 and the resulting recombinant plasmid has been designated pOfACT(1.6). The 1.6-kb pOfACT(1.6) insert hybridizes only to those restriction fragments of the yeast actin gene containing actin coding sequences. When hybridized to macronuclear DNA under conditions that allow the yeast probe to hybridize to all three macronuclear pieces, the pOfACT(1.6) insert hybridizes only to the 1.6-kb piece. Under less stringent conditions the insert also hybridizes to the 1.4-kb piece, but it shows no hybridization to the 1.2-kb DNA. The three macronuclear pieces homologous to the yeast actin gene thus differ in sequence and are interpreted as a related family of actin genes. Each of these pieces could accommodate an actin coding sequence, which in yeast, Dictyostelium discoideum, and Drosophila melanogaster is 1.1 kb, and an additional 0.1-0.5 kb of noncoding DNA.