Molecular components of the archaeal nucleosome

Here we describe the organization of the archaeal nucleosome, in which four archaeal histones are circumscribed by approximately 80 bp of DNA. Through a combination of sequence comparisons, 3D structural studies, site-directed mutagenesis and assays for DNA binding, we have assigned functions to most of the individual residues in the histone fold of the representative archaeal histone rHMfB. By SELEX selection, the sequences of DNA molecules that are most readily bound and wrapped by rHMfB into archaeal nucleosomes in vitro have been identified, and these define DNA structures that position archaeal nucleosome assembly.     

Identification of the archaeal NMN adenylyltransferase gene

Increasing evidence on the importance of fluctuations in NAD+ levels in the living cell is accumulating. Therefore a deeper knowledge on the regulation of coenzyme synthesis and recycling is required. In this context the study of NMN adenylyltransferase (EC 2.7.7. 1), a key enzyme in the NAD+ biosynthetic pathway, assumes a remarkable relevance. We have previously purified to homogeneity and characterized the protein from the thermophilic archaeon Sulfolobus solfataricus. The determination of partial sequence of the S. solfataricus enzyme, together with the recent availability of the genome sequence of the archaeon Methanococcus jannaschii allowed us, based on sequence similarity, to identify the M. jannaschii NMN adenylyltransferase gene. As far as we know from literature, this is the first report on the NMN adenylyltransferase gene.     

RNA channelling by the archaeal exosome

Exosomes are complexes containing 3' --> 5' exoribonucleases that have important roles in processing, decay and quality control of various RNA molecules. Archaeal exosomes consist of a hexameric core of three active RNase PH subunits (ribosomal RNA processing factor (Rrp)41) and three inactive RNase PH subunits (Rrp42). A trimeric ring of subunits with putative RNA-binding domains (Rrp4/cep1 synthetic lethality (Csl)4) is positioned on top of the hexamer on the opposite side to the RNA degrading sites. Here, we present the 1.6 A resolution crystal structure of the nine-subunit exosome of Sulfolobus solfataricus and the 2.3 A structure of this complex bound to an RNA substrate designed to be partly trimmed rather than completely degraded. The RNA binds both at the active site on one side of the molecule and on the opposite side in the narrowest constriction of the central channel. Multiple substrate-binding sites and the entrapment of the substrate in the central channel provide a rationale for the processive degradation of extended RNAs and the stalling of structured RNAs.     

Assembly and function of the archaeal flagellum

Motility is a common behaviour in prokaryotes. Both bacteria and archaea use flagella for swimming motility, but it has been well documented that structures of the flagellum from these two domains of life are completely different, although they contribute to a similar function. Interestingly, information available to date has revealed that structurally archaeal flagella are more similar to bacterial type?IV pili rather than to bacterial flagella. With the increasing genome sequence information and advancement in genetic tools for archaea, identification of the components involved in the assembly of the archaeal flagellum is possible. A subset of these components shows similarities to components from type?IV pilus-assembly systems. Whereas the molecular players involved in assembly of the archaeal flagellum are being identified, the mechanics and dynamics of the assembly of the archaeal flagellum have yet to be established. Recent computational analysis in our laboratory has identified conserved highly charged loop regions within one of the core proteins of the flagellum, the membrane integral protein FlaJ, and predicted that these are involved in the interaction with the assembly ATPase FlaI. Interestingly, considerable variation was found among the loops of FlaJ from the two major subkingdoms of archaea, the Euryarchaeota and the Crenarchaeota. Understanding the assembly pathway and creating an interaction map of the molecular players in the archaeal flagellum will shed light on the details of the assembly and also the evolutionary relationship to the bacterial type?IV pili-assembly systems.     

Molecular dynamics studies of the archaeal translocon

The translocon is a protein-conducting channel conserved over all domains of life that serves to translocate proteins across or into membranes. Although this channel has been well studied for many years, the recent discovery of a high-resolution crystal structure opens up new avenues of exploration. Taking advantage of this, we performed molecular dynamics simulations of the translocon in a fully solvated lipid bilayer, examining the translocation abilities of monomeric SecYEbeta by forcing two helices comprised of different amino acid sequences to cross the channel. The simulations revealed that the so-called plug of SecYEbeta swings open during translocation, closing thereafter. Likewise, it was established that the so-called pore ring region of SecYEbeta forms an elastic, yet tight, seal around the translocating oligopeptides. The closed state of the channel was found to block permeation of all ions and water molecules; in the open state, ions were blocked. Our results suggest that the SecYEbeta monomer is capable of forming an active channel.     

佛斯特拉古菌门（Verstraetearchaeota）研究进展

产甲烷古菌广泛分布在缺氧环境中,是有机质厌氧降解产甲烷过程中的关键功能微生物。它们在全球碳元素循环、气候变化等方面发挥着十分重要的作用。传统观念认为产甲烷古菌仅分布在广古菌门（Euryarchaeota）中,最新研究发现一系列新的非广古菌门（non-Euryarchaeota）产甲烷古菌,推测其不仅具有产甲烷能力,可能还具有发酵复杂有机物的代谢潜力。本文围绕佛斯特拉古菌门（Verstraetearchaeota）产甲烷古菌,系统阐述了它的系统分类、碳代谢机制和生态学分布等方面的研究进展,并展望了未来发展趋势。     

Modelling the evolution of the archaeal tryptophan synthase

Background  

Microorganisms and plants are able to produce tryptophan. Enzymes catalysing the last seven steps of tryptophan biosynthesis are encoded in the canonical trp operon. Among the trp genes are most frequently trpA and trpB, which code for the alpha and beta subunit of tryptophan synthase. In several prokaryotic genomes, two variants of trpB (named trpB1 or trpB2) occur in different combinations. The evolutionary history of these trpB genes is under debate.     

Coexistence of bacterial leucyl-tRNA synthetases with archaeal tRNA binding domains that distinguish tRNALeu in the archaeal mode

Leucyl-tRNA (transfer RNA) synthetase (LeuRS) is a multi-domain enzyme, which is divided into bacterial and archaeal/eukaryotic types. In general, one specific LeuRS, the domains of which are of the same type, exists in a single cell compartment. However, some species, such as the haloalkaliphile Natrialba magadii, encode two cytoplasmic LeuRSs, NmLeuRS1 and NmLeuRS2, which are the first examples of naturally occurring chimeric enzymes with different domains of bacterial and archaeal types. Furthermore, N. magadii encodes typical archaeal tRNA^Leus. The tRNA recognition mode, aminoacylation and translational quality control activities of these two LeuRSs are interesting questions to be addressed. Herein, active NmLeuRS1 and NmLeuRS2 were successfully purified after gene expression in Escherichia coli. Under the optimized aminoacylation conditions, we discovered that they distinguished cognate NmtRNA^Leu in the archaeal mode, whereas the N-terminal region was of the bacterial type. However, NmLeuRS1 exhibited much higher aminoacylation and editing activity than NmLeuRS2, suggesting that NmLeuRS1 is more likely to generate Leu-tRNA^Leu for protein biosynthesis. Moreover, using NmLeuRS1 as a model, we demonstrated misactivation of several non-cognate amino acids, and accuracy of protein synthesis was maintained mainly via post-transfer editing. This comprehensive study of the NmLeuRS/tRNA^Leu system provides a detailed understanding of the coevolution of aminoacyl-tRNA synthetases and tRNA.     

C-terminal domain of archaeal O-phosphoseryl-tRNA kinase displays large-scale motion to bind the 7-bp D-stem of archaeal tRNASec

O-Phosphoseryl-tRNA kinase (PSTK) is the key enzyme in recruiting selenocysteine (Sec) to the genetic code of archaea and eukaryotes. The enzyme phosphorylates Ser-tRNA^Sec to produce O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) that is then converted to Sec-tRNA^Sec by Sep-tRNA:Sec-tRNA synthase. Earlier we reported the structure of the Methanocaldococcus jannaschii PSTK (MjPSTK) complexed with AMPPNP. This study presents the crystal structure (at 2.4-Å resolution) of MjPSTK complexed with an anticodon-stem/loop truncated tRNA^Sec (Mj*tRNA^Sec), a good enzyme substrate. Mj*tRNA^Sec is bound between the enzyme’s C-terminal domain (CTD) and N-terminal kinase domain (NTD) that are connected by a flexible 11 amino acid linker. Upon Mj*tRNA^Sec recognition the CTD undergoes a 62-Å movement to allow proper binding of the 7-bp D-stem. This large reorganization of the PSTK quaternary structure likely provides a means by which the unique tRNA^Sec species can be accurately recognized with high affinity by the translation machinery. However, while the NTD recognizes the tRNA acceptor helix, shortened versions of MjPSTK (representing only 60% of the original size, in which the entire CTD, linker loop and an adjacent NTD helix are missing) are still active in vivo and in vitro, albeit with reduced activity compared to the full-length enzyme.     

The archaeal cell envelope

At first glance, archaea and bacteria look alike; however, the composition of the archaeal cell envelope is fundamentally different from the bacterial cell envelope. With just one exception, all archaea characterized to date have only a single membrane and most are covered by a paracrystalline protein layer. This Review discusses our current knowledge of the composition of the archaeal cell surface. We describe the wide range of cell wall polymers, O- and N-glycosylated extracellular proteins and other cell surface structures that archaea use to interact with their environment.     

The archaeal exosome localizes to the membrane

We studied the cellular localization of the archaeal exosome, an RNA-processing protein complex containing orthologs of the eukaryotic proteins Rrp41, Rrp42, Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. Fractionation of cell-free extracts of Sulfolobus solfataricus in sucrose density gradients revealed that DnaG and the active-site comprising subunit Rrp41 are enriched together with surface layer proteins in a yellow colored ring, implicating that the exosome is membrane-bound. In accordance with this assumption, DnaG and Rrp41 were detected at the periphery of the cell by immunofluorescence microscopy. Our finding suggests that RNA processing in Archaea is spatially organized.

Structured summary

MINT-7891213: Rrp41 (uniprotkb:Q9UXC2) and DnaG (uniprotkb:P95980) colocalize (MI:0403) by cosedimentation in solution (MI:0028)MINT-7891235: Rrp41 (uniprotkb:Q9UXC2), DnaG (uniprotkb:P95980) and SlaA (uniprotkb:Q2M1E7) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-7891278: Rrp41 (uniprotkb:Q9UXC2) and DnaG (uniprotkb:P95980) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

The archaeal origins of the eukaryotic translational system

Among the 78 eukaryotic ribosomal proteins, eleven are specific to Eukarya, 33 are common only to Archaea and Eukarya and 34 are homologous (at least in part) to those of both Bacteria and Archaea. Several other translational proteins are common only to Eukarya and Archaea (e.g., IF2a, SRP19, etc.), whereas others are shared by the three phyla (e.g., EFTu/EF1A and SRP54). Although this and other analyses strongly support an archaeal origin for a substantial fraction of the eukaryotic translational machinery, especially the ribosomal proteins, there have been numerous unique and ubiquitous additions to the eukaryotic translational system besides the 11 unique eukaryotic ribosomal proteins. These include peptide additions to most of the 67 archaeal homolog proteins, rRNA insertions, the 5.8S RNA and the Alu extension to the SRP RNA. Our comparative analysis of these and other eukaryotic features among the three different cellular phylodomains supports the idea that an archaeal translational system was most likely incorporated by means of endosymbiosis into a host cell that was neither bacterial nor archaeal in any modern sense. Phylogenetic analyses provide support for the timing of this acquisition coinciding with an ancient bottleneck in prokaryotic diversity.     

Indicators from archaeal secretomes

Just as in the Eukarya and the Bacteria, members of the Archaea need to export proteins beyond the cell membrane. This would be required to fulfill a variety of essential functions such as nutrient acquisition and biotransformations, maintenance of extracellular structures and more. Apart from the Eukarya and the Bacteria however, members of the Archaea share a number of unique characteristics. Does this uniqueness extend to the protein secretion system? It was the objective of this study to answer this question. To overcome the limited experimental information on secreted proteins in Archaea, this study was carried out by subjecting the available archaeal genomes, which represent halophiles, thermophiles, and extreme thermophiles, to bioinformatics analysis. Specifically, to examine the properties of the secretomes of the Archaea using the ExProt program. A total of 24 genomes were analyzed. Secretomes were found to fall in the range of 6% of total ORFs (Methanopyrus kandleri) to 19% (Halobacterium sp. NRC-1). Methanosarcina acetivorans has the highest fraction of lipoproteins (at 89) and the lowest (at 1) were members of the Thermoplasma, Pyrobaculum aerophilum, and Nanoarchaeum equitans. Based on the Tat consensus sequence, contribution of these secreted proteins to the secretomes were negligible, making up 8 proteins out of a total of 7105 predicted exported proteins. Amino acid composition, an attribute of signal peptides not used as a selection criteria by ExProt, of predicted archaeal signal peptides show that in the haloarchaea secretomes, the frequency of the amino acid Lys is much lower than that seen in bacterial signal peptides, but is compensated for by a higher frequency of Arg. It also showed that higher frequencies for Thr, Val, and Gly contribute to the hydrophobic character in haloarchaeal signal peptides, unlike bacterial signal peptides in which the hydrophobic character is dominated by Leu and Ile.     

The archaeal PCNA proteins

PCNA (proliferating-cell nuclear antigen) is a ring-shaped protein that encircles duplex DNA and plays an essential role in many DNA metabolic processes. The PCNA protein interacts with a large number of cellular factors and modulates their enzymatic activities. In the present paper, we summarize the structures, functions and interactions of the archaeal PCNA proteins.     

Multi-scale architecture of archaeal chromosomes

1. Download : Download high-res image (109KB)
2. Download : Download full-size image

     

Biotechnological uses of archaeal extremozymes

Archaea have developed a variety of molecular strategies to survive the often harsh environments in which they exist. Although the rules that allow archaeal enzymes to fulfill their catalytic functions under extremes of salinity, temperature or pressure are not completely understood, the stability of these extremophilic enzymes, or extremozymes, in the face of adverse conditions has led to their use in a variety of biotechnological applications in which such tolerances are advantageous. In the following, examples of commercially important archaeal extremozymes are presented, potentially useful archaeal extremozyme sources are identified and solutions to obstacles currently hindering wider use of archaeal extremozymes are discussed.     

Protein-protein interactions in the archaeal core replisome

Most of the core components of the archaeal chromosomal DNA replication apparatus share significant protein sequence similarity with eukaryotic replication factors, making the Archaea an excellent model system for understanding the biology of chromosome replication in eukaryotes. The present review summarizes current knowledge of how the core components of the archaeal chromosome replication apparatus interact with one another to perform their essential functions.