Charles J. Daniels

Gene structure, organization, and expression in archaebacteria.

Major advances have recently been made in understanding the molecular biology of the archaebacteria. In this review, we compare the structure of protein and stable RNA-encoding genes cloned and sequenced from each of the major classes of archaebacteria: the methanogens, extreme halophiles, and acid thermophiles. Protein-encoding genes, including some encoding proteins directly involved in methanogenesis and photoautotrophy, are analyzed on the basis of gene organization and structure, transcriptional control signals, codon usage, and evolutionary conservation. Stable RNA-encoding genes are compared for gene organization and structure, transcriptional signals, and processing events involved in RNA maturation, including intron removal. Comparisons of archaebacterial structures and regulatory systems are made with their eubacterial and eukaryotic homologs.

The Complete Genome Sequence of Haloferax volcanii DS2, a Model Archaeon

Background Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general. Methodology/Principal Findings We report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively) and the pHV2 plasmid (6.4 kb). Conclusions/Significance The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.

STRUCTURE AND FUNCTION OF ARCHAEAL BOX C/D SRNP CORE PROTEINS

Nop56p and Nop58p are two core proteins of the box C/D snoRNPs that interact concurrently with fibrillarin and snoRNAs to function in enzyme assembly and catalysis. Here we report the 2.9 Å resolution co-crystal structure of an archaeal homolog of Nop56p/Nop58p, Nop5p, in complex with fibrillarin from Archaeoglobus fulgidus (AF) and the methyl donor S-adenosyl-L-methionine. The N-terminal domain of Nop5p forms a complementary surface to fibrillarin that serves to anchor the catalytic subunit and to stabilize cofactor binding. A coiled coil in Nop5p mediates dimerization of two fibrillarin–Nop5p heterodimers for optimal interactions with bipartite box C/D RNAs. Structural analysis and complementary biochemical data demonstrate that the conserved C-terminal domain of Nop5p harbors RNA-binding sites. A model of box C/D snoRNP assembly is proposed based on the presented structural and biochemical data.

Is gene expression in Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors?

Sir, Gene expression is regulated by different mechanisms in different organisms. The bacterial core RNA polymerase (a2bb 0) discriminates between subsets of promoters by binding different s factors. Eukaryotes have evolved a more complicated system making use of three RNA polymerases to direct synthesis from different promoter families. Archaea possess a simplified version of RNA polymerase II transcription machinery with a single multisubunit RNA polymerase and a subset, TBP and TFIIB, of general transcription factors (Reeve et al., 1997, Cell 89: 999±1002). However, multiple transcription factor homologues have been identified in several archaea including Halobacterium NRC-1 (Ng et al., 1998, Genome Res 8: 1131±1141), Haloferax volcanii (Thompson et al., 1999, Mol Microbiol 33: 1081±1092) and Pyrococcus horikoshii OT3 (Kawarabayasi et al., 1998, DNA Res 5: 147±155). With the impending completion of the Halobacterium NRC-1 genome project, this extreme halophile is turning out to be a champion of multiple transcription factors, with six tbp and seven tfb genes (http://zdna.micro. umass.edu/haloweb).

Properties of H. volcanii tRNA Intron Endonuclease Reveal a Relationship between the Archaeal and Eucaryal tRNA Intron Processing Systems

To better understand the relationship between archaeal and eucaryal tRNA introns and their processing systems, we have cloned the gene encoding the tRNA intron endonucleases from the archaeon H. volcanii. The gene encodes a 37 kDa protein that appears to be present as a homodimer under native conditions. Recombinant forms of this protein were expressed in E. coli and found to cleave precursor tRNAs lacking full mature tRNA structure, a property observed for the native endonuclease. Comparative sequence analysis revealed that similar proteins existed in other Archaea and that these proteins have significant similarity with two subunits of the yeast tRNA intron endonuclease. These results provide evidence that the archaeal and eucaryal tRNA intron processing systems are related and suggest a common origin for tRNA introns in these organisms.

Expression and heat-responsive regulation of a TFIIB homologue from the archaeon Haloferax volcanii.

Multiple divergent genes encoding the eukaryal‐like TFIIB (TFB) transcription initiation factor have been identified in the archaeon Haloferax volcanii. Expression of one of these TFB‐encoding genes, referred to here as tfb2, was induced specifically in response to heat shock at the transcription level. A time course for tfb2 induction demonstrated that mRNA levels increased as much as eightfold after 15 min at 60°C. A transcription fusion of the tfb2 promoter region with a stable RNA reporter gene confirmed the heat responsiveness of the tfb2 core promoter, and immunoblot analysis using antibodies generated against a recombinant His‐tagged TFB2 showed that the protein levels of one TFB increased slightly in response to elevated temperatures. An archaeal consensus TATA element (5′‐TTTATA‐3′) was located 110 bp upstream of the translation start site and appeared to be used for both basal and heat shock‐induced expression. The long DNA leader region (79 bp) preceding the predicted AUG translation start codon for TFB2 contained a T‐rich sequence element located 22 bp downstream of the transcription start site. Using an in vivo transcription termination assay, we demonstrated that this T‐rich element can function as a sequence‐dependent transcription terminator, which may serve to downregulate expression of the tfb2 gene under both non‐heat shock and heat shock conditions.

Heat shock inducibility of an archaeal TATA-like promoter is controlled by adjacent sequence elements

The expression of a heat‐inducible cct1 (chaperonin‐containing Tcp‐1) family member gene is regulated at the transcription level in the archaeon Haloferax volcanii. Transcriptional fusions of the cct1 promoter region with a yeast proline tRNA reporter gene were constructed to analyse the functional domains of this archaeal heat shock promoter. Both basal and heat‐induced transcription of the reporter gene was directed by an archaeal consensus TATA element (5′‐TTTATA‐3′) centred 25 bp upstream of the transcription start site. Deletion mutagenesis indicated that the 5′ boundary of the cct1 regulatory region mapped to position − 37. Nucleotide alignment with the 5′ flanking regions of two additional cct‐related genes identified in H. volcanii showed a high degree of sequence conservation between positions +1 and − 37, especially in and immediately surrounding the TATA element of the putative core promoter. Mutational analysis of conserved sequences demonstrated that basal and heat‐induced transcription required sequence elements located upstream and downstream of the TATA‐box. These findings indicate that the regulatory sequences involved in heat‐induced transcription lie within the core promoter region and suggest that the mechanism controlling heat shock gene expression in H. volcanii differs from the bacterial and eukaryal strategies.

Shotgun proteomics of the haloarchaeon Haloferax volcanii.

Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.

Splicing of intron-containing tRNATrp by the archaeon Haloferax volcanii occurs independent of mature tRNA structure.

We have investigated the requirements for mature tRNA structure in the in vivo splicing of theHaloferax volcanii, intron-containing tRNATrp RNA. A partial tRNATrp gene, which contained only the anticodon stem-loop region of the mature tRNA, was fused to a carrier yeast tRNA gene for expression in H. volcanii. Transcripts from this hybrid gene were found to be processed by endonuclease and ligase at the tRNATrp exon-intron boundaries. These results verify that the substrate recognition properties of the halobacterial endonuclease observed in vitro reflect the properties of this enzymein vivo, namely that mature tRNA structure is not essential for recognition by the endonuclease. The independence of these reactions on mature tRNA provides further support for a relationship between archaeal tRNA and rRNA intron-processing systems and highlight a difference in the substrate recognition properties between the archaeal and eucaryal processing systems. The significance of these differences is discussed in light of the observation that the tRNA endonucleases of these organisms are related.

In vivo processing of an intron-containing archael tRNA.

In vitro studies on the processing of halobacterial tRNA introns have led to the proposal that archaeal and eukaryotic tRNA intron endonucleases have distinctly different requirements for the recognition of pre‐tRNAs. Using a Haloferax volcanii in vivo expression vector we have examined the in vivo processing of modified forms of the halobacterial intron‐containing tRNATrp gene. As observed in vitro, changes in the exon–intron boundary structure of this pre‐tRNA block processing. Intron sequences, other than those at the exon–intron boundaries, are not essential for processing in vivo. We also show that conversion of the tryptophan anticodon to an opal suppressor anticodon is tolerated when the exon‐intron boundary structure is maintained.