Both kinds of RNase P in all domains of life: surprises galore.

RNase P, an essential housekeeping endonuclease needed for 5'-processing of tRNAs, exists in two distinct forms: one with an RNA- and the other with a protein-based active site. The notion that the protein form of RNase P exists only in eukaryotes has been upended by the recent discovery of a protein-only variant in Bacteria and Archaea. The use of these two divergent scaffolds, shaped by convergent evolution, in all three domains of life inspires questions relating to the ancestral form of RNase P, as well as their origins and function(s) in vivo. Results from our analysis of publicly available bacterial and archaeal genomes suggest that the widespread RNA-based ribonucleoprotein variant is likely the ancient form. We also discuss the possible genetic origins and function of RNase P, including how the simultaneous presence of its variants may contribute to the fitness of their host organisms.

Complete Genome Sequences of 44 Arthrobacter Phages.

We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae, Myoviridae, and Podoviridae) are represented.

Sequence Analysis and Comparative Study of the Protein Subunits of Archaeal RNase P.

RNase P, a ribozyme-based ribonucleoprotein (RNP) complex that catalyzes tRNA 5'-maturation, is ubiquitous in all domains of life, but the evolution of its protein components (RNase P proteins, RPPs) is not well understood. Archaeal RPPs may provide clues on how the complex evolved from an ancient ribozyme to an RNP with multiple archaeal and eukaryotic (homologous) RPPs, which are unrelated to the single bacterial RPP. Here, we analyzed the sequence and structure of archaeal RPPs from over 600 available genomes. All five RPPs are found in eight archaeal phyla, suggesting that these RPPs arose early in archaeal evolutionary history. The putative ancestral genomic loci of archaeal RPPs include genes encoding several members of ribosome, exosome, and proteasome complexes, which may indicate coevolution/coordinate regulation of RNase P with other core cellular machineries. Despite being ancient, RPPs generally lack sequence conservation compared to other universal proteins. By analyzing the relative frequency of residues at every position in the context of the high-resolution structures of each of the RPPs (either alone or as functional binary complexes), we suggest residues for mutational analysis that may help uncover structure-function relationships in RPPs.

Assigning a function to a conserved archaeal metallo-ß-lactamase from Haloferax volcanii.

The metallo-ß-lactamase family of enzymes comprises a large group of proteins with diverse functions in the metabolism of the cell. Among others, this superfamily contains proteins which are involved in DNA and RNA metabolism, acting as nucleases in e.g. repair and maturation. Many proteins have been annotated in prokaryotic genomes as being potential metallo-ß-lactamases, but very often the function has not been proven. The protein HVO_2763 from Haloferax volcanii is such a potential metallo-ß-lactamase. HVO_2763 has sequence similarity to the metallo-ß-lactamase tRNase Z, a tRNA 3' processing endonuclease. Here, we report the characterisation of this metallo-ß-lactamase HVO_2763 in the halophilic archaeon Haloferax volcanii. Using different in vitro assays with the recombinant HVO_2763, we could show that the protein does not have tRNA 3' processing or exonuclease activity. According to transcriptome analyses of the HVO_2763 deletion strain, expression of proteins involved in membrane transport is downregulated in the mutant. Therefore, HVO_2763 might be involved directly or indirectly in membrane transport.

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.

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 A 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.

PACRAT: a database and analysis system for archaeal and bacterial intergenic sequence features.

Analysis of intergenic sequences for purposes such as the investigation of transcriptional signals or the identification of small RNA genes is frequently complicated by traditional biological database structures. Genome data is commonly treated as chromosome-length sequence records, detailed by gene calls demarcating subsequences of the chromosomes. Given this model, the determination of non-called subsequences between any gene and its nearest neighbors requires an exhaustive search of all gene calls associated with the chromosome. Further compounding the issue, the location of intergenic regions for many called genes cannot be resolved unambiguously due to uncertainties in gene boundaries, as well as the presence of other conflicting gene calls. To address these difficulties we have constructed the PACRAT (http://www.biosci.ohio-state.edu/~pacrat/) database system. PACRAT preprocesses GenBank genome submissions, evaluates for every gene the character of its relationship to those genes nearest to it, and produces a relationally linked model of the gene ordering for the genome. Using this information, the interface allows the researcher to query gene data as well as intergenic sequence data based on a number of criteria. These include the ability to filter searches based on the status of start and stop positions, or upstream/downstream sequences as conflicting with called genes and automated extension of upstream or downstream searches to find probable operon promoters or terminators. The database is also indexed by KEGG classification, allowing, for example, functionally-related groups of high-quality promoter-containing regions to be easily retrieved as a group.