The combined DNA and RNA synthetic capabilities of archaeal DNA primase facilitate primer hand-off to the replicative DNA polymerase.

Replicative DNA polymerases cannot initiate DNA synthesis de novo and rely on dedicated RNA polymerases, primases, to generate a short primer. This primer is then extended by the DNA polymerase. In diverse archaeal species, the primase has long been known to have the ability to synthesize both RNA and DNA. However, the relevance of these dual nucleic acid synthetic modes for productive primer synthesis has remained enigmatic. In the current work, we reveal that the ability of primase to polymerize DNA serves dual roles in promoting the hand-off of the primer to the replicative DNA polymerase holoenzyme. First, it creates a 5'-RNA-DNA-3' hybrid primer which serves as an optimal substrate for elongation by the replicative DNA polymerase. Second, it promotes primer release by primase. Furthermore, modeling and experimental data indicate that primase incorporates a deoxyribonucleotide stochastically during elongation and that this switches the primase into a dedicated DNA synthetic mode polymerase.

Approaches to study CRISPR RNA biogenesis and the key players involved.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide an inheritable and adaptive immune system against phages and foreign genetic elements in many bacteria and archaea. The three stages of CRISPR-Cas immunity comprise adaptation, CRISPR RNA (crRNA) biogenesis and interference. The maturation of the pre-crRNA into mature crRNAs, short guide RNAs that target invading nucleic acids, is crucial for the functionality of CRISPR-Cas defense systems. Mature crRNAs assemble with Cas proteins into the ribonucleoprotein (RNP) effector complex and guide the Cas nucleases to the cognate foreign DNA or RNA target. Experimental approaches to characterize these crRNAs, the specific steps toward their maturation and the involved factors, include RNA-seq analyses, enzyme assays, methods such as cryo-electron microscopy, the crystallization of proteins, or UV-induced protein-RNA crosslinking coupled to mass spectrometry analysis. Complex and multiple interactions exist between CRISPR-cas-encoded specific riboendonucleases such as Cas6, Cas5d and Csf5, endonucleases with dual functions in maturation and interference such as the enzymes of the Cas12 and Cas13 families, and nucleases belonging to the cell's degradosome such as RNase E, PNPase and RNase J, both in the maturation as well as in interference. The results of these studies have yielded a picture of unprecedented diversity of sequences, enzymes and biochemical mechanisms.

Contribution of protein Gar1 to the RNA-guided and RNA-independent rRNA:Ψ-synthase activities of the archaeal Cbf5 protein.

Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ2603 in Pyrococcus abyssi 23S rRNA and of Ψ55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ2607 (Ψ2603 in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.

Structure and function of the archaeal exosome.

The RNA-degrading exosome in archaea is structurally very similar to the nine-subunit core of the essential eukaryotic exosome and to bacterial polynucleotide phosphorylase (PNPase). In contrast to the eukaryotic exosome, PNPase and the archaeal exosome exhibit metal ion-dependent, phosphorolytic activities and synthesize heteropolymeric RNA tails in addition to the exoribonucleolytic RNA degradation in 3' → 5' direction. The archaeal nine-subunit exosome consists of four orthologs of eukaryotic exosomal subunits: the RNase PH-domain-containing subunits Rrp41 and Rrp42 form a hexameric ring with three active sites, whereas the S1-domain-containing subunits Rrp4 and Csl4 form an RNA-binding trimeric cap on the top of the ring. In vivo, this cap contains Rrp4 and Csl4 in variable amounts. Rrp4 confers poly(A) specificity to the exosome, whereas Csl4 is involved in the interaction with the archaea-specific subunit of the complex, the homolog of the bacterial primase DnaG. The archaeal DnaG is a highly conserved protein and its gene is present in all sequenced archaeal genomes, although the exosome was lost in halophilic archaea and some methanogens. In exosome-containing archaea, DnaG is tightly associated with the exosome. It functions as an additional RNA-binding subunit with poly(A) specificity in the reconstituted exosome of Sulfolobus solfataricus and enhances the degradation of adenine-rich transcripts in vitro. Not only the RNA-binding cap but also the hexameric Rrp41-Rrp42 ring alone shows substrate selectivity and prefers purines over pyrimidines. This implies a coevolution of the exosome and its RNA substrates resulting in 3'-ends with different affinities to the exosome.

High-purity enzymatic synthesis of site-specifically modified tRNA.

Transfer RNA (tRNA) molecules play the key role in adapting the genetic code sequences with amino acids. The execution of this key role is highly dependent on the presence of modified nucleotides in tRNA, each of which performs a distinct function. To better understand how individual modifications modulate tRNA function, a method to isolate and purify a site-specifically modified tRNA is essential. This chapter describes an enzymatic method to synthesize a site-specifically modified tRNA, followed by purification of this tRNA away from unmodified tRNA using a selective oligonucleotide-based hybridization approach. This method is broadly applicable to site-specific tRNA modifications that interfere with nucleic-acid base-pairing principles.

Biosynthesis of 4-thiouridine in tRNA in the methanogenic archaeon Methanococcus maripaludis.

4-Thiouridine (s(4)U) is a conserved modified nucleotide at position 8 of bacterial and archaeal tRNAs and plays a role in protecting cells from near-UV killing. Escherichia coli employs the following two enzymes for its synthesis: the cysteine desulfurase IscS, which forms a Cys persulfide enzyme adduct from free Cys; and ThiI, which adenylates U8 and transfers sulfur from IscS to form s(4)U. The C-terminal rhodanese-like domain (RLD) of ThiI is responsible for the sulfurtransferase activity. The mechanism of s(4)U biosynthesis in archaea is not known as many archaea lack cysteine desulfurase and an RLD of the putative ThiI. Using the methanogenic archaeon Methanococcus maripaludis, we show that deletion of ThiI (MMP1354) abolished the biosynthesis of s(4)U but not of thiamine. MMP1354 complements an Escherichia coli ΔthiI mutant for s(4)U formation, indicating that MMP1354 is sufficient for sulfur incorporation into s(4)U. In the absence of an RLD, MMP1354 uses Cys(265) and Cys(268) located in the PP-loop pyrophosphatase domain to generate persulfide and disulfide intermediates for sulfur transfer. In vitro assays suggest that S(2-) is a physiologically relevant sulfur donor for s(4)U formation catalyzed by MMP1354 (K(m) for Na(2)S is ∼1 mm). Thus, methanogenic archaea developed a strategy for sulfur incorporation into s(4)U that differs from bacteria; this may be an adaptation to life in sulfide-rich environments.

RNA-guided genetic silencing systems in bacteria and archaea.

Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.

Expression profiles and physiological roles of two types of molecular chaperonins from the hyperthermophilic archaeon Thermococcus kodakarensis.

Thermococcus kodakarensis possesses two chaperonins, CpkA and CpkB, and their expression is induced by the downshift and upshift, respectively, of the cell cultivation temperature. The expression levels of the chaperonins were examined by using specific antibodies at various cell growth temperatures in the logarithmic and stationary phases. At 60 degrees C, CpkA was highly expressed in both the logarithmic and stationary phases; however, CpkB was not expressed in either phase. At 85 degrees C, CpkA and CpkB were expressed in both phases; however, the CpkA level was decreased in the stationary phase. At 93 degrees C, CpkA was expressed only in the logarithmic phase and not in the stationary phase. In contrast, CpkB was highly expressed in both phases. The results of reverse transcription-PCR experiments showed the same growth phase- and temperature-dependent profiles as observed in immunoblot analyses, indicating that the expression of cpkA and cpkB is regulated at the mRNA level. The cpkA or cpkB gene disruptant was then constructed, and its growth profile was monitored. The cpkA disruptant showed poor cell growth at 60 degrees C but no significant defects at 85 degrees C and 93 degrees C. On the other hand, cpkB disruption led to growth defects at 93 degrees C but no significant defects at 60 degrees C and 85 degrees C. These data indicate that CpkA and CpkB are necessary for cell growth at lower and higher temperatures, respectively. The logarithmic-phase-dependent expression of CpkA at 93 degrees C suggested that CpkA participates in initial cell growth in addition to lower-temperature adaptation. Promoter mapping and quantitative analyses using the Phr (Pyrococcus heat-shock regulator) gene disruptant revealed that temperature-dependent expression was achieved in a Phr-independent manner.

Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene.

A system where archaeal gene expression could be controlled by simple manipulation of growth conditions would enable the construction of conditional lethal mutants in essential genes, and permit the controlled overproduction of proteins in their native host. As tools for the genetic manipulation of Haloferax volcanii are well developed, we set out to identify promoters with a wide dynamic range of expression in this organism. Tryptophan is the most costly amino acid for the cell to make, so we reasoned that tryptophan-regulated promoters might be good candidates. Microarray analysis of H. volcanii gene expression in the presence and absence of tryptophan identified a tryptophanase gene (tna) that showed strong induction in the presence of tryptophan. qRT-PCR revealed a very fast response and an up to 100-fold induction after tryptophan addition. This result has been confirmed using three independent reporter genes (cct1, pyrE2 and bgaH). Vectors containing this promoter will be very useful for investigating gene function in H. volcanii and potentially in other halophilic archaea. To demonstrate this, we used the promoter to follow the consequences of depletion of the essential chaperonin protein CCT1, and to determine the ability of heterologous CCT proteins to function in H. volcanii.

Three 2-oxoacid dehydrogenase operons in Haloferax volcanii: expression, deletion mutants and evolution.

Two unrelated protein families catalyse the oxidative decarboxylation of 2-oxoacids, i.e. the 2-oxoacid dehydrogenase complexes (OADHCs) and the 2-oxoacid ferredoxin oxidoreductases (OAFORs). In halophilic archaea, OAFORs were found to be responsible for decarboxylation of pyruvate and 2-oxoglutarate. Nevertheless, two gene clusters encoding OADHCs were found previously in Haloferax volcanii, but their biological function remained obscure. Here a third oadhc gene cluster of H. volcanii is presented. To characterize the function, the genes encoding the E1 subunit were inactivated in all three gene clusters by in-frame deletions. Under aerobic conditions none of the three mutants showed any phenotypic difference from the wild-type in various media. However, growth yields of two mutants were considerably lower than that of wild-type under nitrate-respirative conditions in complex medium. Northern blot analyses revealed (1) that polycistronic transcripts are formed and all three gene clusters are bona fide operons and (2) that transcription of all three operons is induced under anaerobic conditions compared to aerobic conditions. Taken together, the three H. volcanii enzymes do not fulfil one of the 'usual' aerobic functions of typical OADHCs, but decarboxylate an as-yet-unidentified novel substrate under anaerobic conditions. A survey of all 28 fully sequenced archaeal genomes revealed that nearly all archaea contain several OAFORs (three to four on average), suggesting that this protein family was already present in their last common ancestor. In contrast, only nine archaea encode one or two OADHCs, indicating that this protein family entered archaea by lateral transfer of the cognate genes from bacteria. This view is underscored by a phylogenetic tree of 33 archaeal and bacterial OADHCs.

MarR-like transcriptional regulator involved in detoxification of aromatic compounds in Sulfolobus solfataricus.

A DNA binding protein, BldR, was identified in the crenarchaeon Sulfolobus solfataricus as a protein 5- to 10-fold more abundant in cells grown in the presence of toxic aldehydes; it binds to regulatory sequences located upstream of an alcohol dehydrogenase gene (Sso2536). BldR is homologous to bacterial representatives of the MarR (multiple antibiotic resistance) family of transcriptional regulators that mediate response to multiple environmental stresses. Transcriptional analysis revealed that the bldR gene was transcribed in a bicistronic unit composed of the genes encoding the transcriptional regulator (Sso1352) and a putative multidrug transporter (Sso1351) upstream. By homology to bacterial counterparts, the bicistron was named the mar-like operon. The level of mar-like operon expression was found to be increased at least 10-fold in response to chemical stress by aromatic aldehydes. Under the same growth conditions, similar enhanced in vivo levels of Sso2536 gene transcript were also measured. The gene encoding BldR was expressed in E. coli, and the recombinant protein was purified to homogeneity. DNA binding assays demonstrated that the protein is indeed a transcription factor able to recognize site specifically both the Sso2536 and mar-like promoters at sites containing palindromic consensus sequences. Benzaldehyde, the substrate of ADH(Ss), stimulates DNA binding of BldR at both promoters. The role of BldR in the auto-activation as well as in the regulation of the Sso2536 gene, together with results of increased operon and gene expression under conditions of exposure to aromatic aldehydes, indicates a novel coordinate regulatory mechanism in cell defense against stress by aromatic compounds.

Transcriptional analysis of the genetic element pSSVx: differential and temporal regulation of gene expression reveals correlation between transcription and replication.

pSSVx from Sulfolobus islandicus strain REY15/4 is a hybrid between a plasmid and a fusellovirus. A systematic study performed by a combination of Northern blot analysis, primer extension, and reverse transcriptase PCR revealed the presence of nine major transcripts whose expression was differentially and temporally regulated over the growth cycle of S. islandicus. The map positions of the RNAs as well as the clockwise and the anticlockwise directions of their transcription were determined. Some genes were clustered and appeared to be transcribed as polycistronic messengers, among which one long transcriptional unit comprised the genes for the plasmid copy number control protein ORF60 (CopG), ORF91, and the replication protein ORF892 (RepA). We propose that a termination readthrough mechanism might be responsible for the formation of more than one RNA species from a single 5' end and therefore that the nine different RNAs corresponded to only seven different transcriptional starts. Three transcripts, ORF76 and two antisense RNAs, countertranscribed RNA1 (ctRNA1) and ctRNA2, were found to be specifically expressed during (and hence correlated to) the phase in which the pSSVx copy number is kept under stringent control, as they were completely switched off upon the onset of the induction of replication.

A story with a good ending: tRNA 3'-end maturation by CCA-adding enzymes.

CCA-adding enzymes (tRNA nucleotidyltransferases) are responsible for the maturation or repair of the functional 3' end of tRNAs. These enzymes are remarkable because they polymerize the essential nucleotides CCA onto the 3' terminus of tRNA precursors without using a nucleic acid template. Recent crystal structures, plus three decades of enzymology, have revealed the elegant mechanisms by which CCA-adding enzymes achieve their substrate specificity in a nucleic acid template independent fashion. The class I CCA-adding enzyme employs both an arginine sidechain and backbone phosphates of the bound tRNA to recognize incoming nucleotides. It switches from C to A addition through changes in the size and shape of the nucleotide-binding pocket, which is progressively altered by the elongating 3' terminus of the tRNA. By contrast, the class II CCA-adding enzyme uses only amino acid sidechains, which form a protein template for incoming nucleotide selection.

Two different mechanisms for tRNA ribose methylation in Archaea: a short survey.

The biogenesis of tRNA involves multiple reactions including post-transcriptional modifications and pre-tRNA splicing. Among the three domains of life, only Archaea have two different mechanisms for tRNA ribose methylation: site-specific 2'-O-methyltransferases and C/D guided-RNA machinery. Recently, the first archaeal tRNA 2'-O-methyltransferase, aTrm56, has been characterized. This enzyme is found in all archaeal genomes sequenced so far except one and belongs to the SPOUT family (class IV) of RNA methyltransferases. Its substrate is the conserved C56 in the T-loop of archaeal tRNAs. In the crenarchaeon Pyrobaculum aerophylum, in which no homologue of this methyltransferase is found, a box C/D guide sRNP insures the ribose methylation of C56. Moreover, a new twist on tRNA processing is the finding, in most euryarchaeal tRNAtrp genes, of a box C/D guide RNA within their intron specifying methylation at two sites. Modification of tRNA is an integral part of the complex maturation process of primary tRNA transcripts. In addition to their role in modification, both modification enzymes and C/D guide RNPs may have a chaperone function insuring the precise folding of the mature, functional tRNA.

Distinct domain functions regulating de novo DNA synthesis of thermostable DNA primase from hyperthermophile Pyrococcus horikoshii.

DNA primases are essential components of the DNA replication apparatus in every organism. Reported here are the biochemical characteristics of a thermostable DNA primase from the thermophilic archaeon Pyrococcus horikoshii, which formed the oligomeric unit L(1)S(1) and synthesized long DNA primers 10 times more effectively than RNA primers. The N-terminal (25KL) and C-terminal halves (20KL) of the large subunit (L) play distinct roles in regulating de novo DNA synthesis of the small catalytic subunit (S). The 25KL domain has a dual function. One function is to depress the large affinity of the intrasubunit domain 20KL for the template DNA until complex (L(1)S(1) unit) formation. The other function is to tether the L subunit tightly to the S subunit, probably to promote effective interaction between the intrasubunit domain 20KL and the active center of the S subunit. The 20KL domain is a central factor to enhance the de novo DNA synthesis activity of the catalytic S subunit since the total affinity of the L(1)S(1) unit is mainly derived from the affinity of 20KL, which is elevated more than 10 times by the heterodimer formation, presumably due to the cancellation of the inhibitory activity of 25KL through tight binding to the S subunit.

Stability of mRNA in the hyperthermophilic archaeon Sulfolobus solfataricus.

Archaea-like bacteria are prokaryotes but, in contrast, use eukaryotic-like systems for key aspects of DNA, RNA, and protein metabolism. mRNA is typically unstable in bacteria and stable in eukaryotes, but little information is available about mRNA half-lives in archaea. Because archaea are generally insensitive to antibiotics, examination of mRNA stability in the hyperthermophile, Sulfolobus solfataricus, required the identification of transcription inhibitors for half-life determinations. An improved lacS promoter-dependent in vitro transcription system was used to assess inhibitor action. Efficient inhibitors were distinguished as blocking both lacSp transcription in vitro and the incorporation of 3H-uracil into bulk RNA in vivo. Actinomycin D was the most stable and potent compound identified. A survey of transcript chemical half-lives normalized to levels of the signal recognition particle 7S RNA ranged from at least 2 h for tfb1, a transcription factor TFIIB paralog, to a minimum of 6.3 min for gln1, one of three glutamine synthetase paralogs. Transcript half-lives for other mRNAs were: 2 h, superoxide dismutase (sod); 37.5 min, glucose dehydrogenase (dhg1); 25 min, alpha-glucosidase (malA); and 13.5 min, transcription factor TFIIB-2 (tfb2) resulting in a minimum average half-life of 54 min. These are the first mRNA half-lives reported for a hyperthermophile or member of the crenarchaea. The unexpected stability of several transcripts has important implications for gene expression and mRNA degradation in this organism.

Cell-free transcription at 95 degrees: thermostability of transcriptional components and DNA topology requirements of Pyrococcus transcription.

Cell-free transcription of archaeal promoters is mediated by two archaeal transcription factors, aTBP and TFB, which are orthologues of the eukaryotic transcription factors TBP and TFIIB. Using the cell-free transcription system described for the hyperthermophilic Archaeon Pyrococcus furiosus by Hethke et al., the temperature limits and template topology requirements of archaeal transcription were investigated. aTBP activity was not affected after incubation for 1 hr at 100 degrees. In contrast, the half-life of RNA polymerase activity was 23 min and that of TFB activity was 3 min. The half-life of a 328-nt RNA product was 10 min at 100 degrees. Best stability of RNA was observed at pH 6, at 400 mm K-glutamate in the absence of Mg(2+) ions. Physiological concentrations of K-glutamate were found to stabilize protein components in addition, indicating that salt is an important extrinsic factor contributing to thermostability. Both RNA and proteins were stabilized by the osmolyte betaine at a concentration of 1 m. The highest activity for RNA synthesis at 95 degrees was obtained in the presence of 1 m betaine and 400 mm K-glutamate. Positively supercoiled DNA, which was found to exist in Pyrococcus cells, can be transcribed in vitro both at 70 degrees and 90 degrees. However, negatively supercoiled DNA was the preferred template at all temperatures tested. Analyses of transcripts from plasmid topoisomers harboring the glutamate dehydrogenase promoter and of transcription reactions conducted in the presence of reverse gyrase indicate that positive supercoiling of DNA inhibits transcription from this promoter.