Landscape of RNA pseudouridylation in archaeon Sulfolobus islandicus.

Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs.

The archaeal Lsm protein from Pyrococcus furiosus binds co-transcriptionally to poly(U)-rich target RNAs.

Posttranscriptional processes in Bacteria include the association of small regulatory RNAs (sRNA) with a target mRNA. The sRNA/mRNA annealing process is often mediated by an RNA chaperone called Hfq. The functional role of bacterial and eukaryotic Lsm proteins is partially understood, whereas knowledge about archaeal Lsm proteins is scarce. Here, we used the genetically tractable archaeal hyperthermophile Pyrococcus furiosus to identify the protein interaction partners of the archaeal Sm-like proteins (PfuSmAP1) using mass spectrometry and performed a transcriptome-wide binding site analysis of PfuSmAP1. Most of the protein interaction partners we found are part of the RNA homoeostasis network in Archaea including ribosomal proteins, the exosome, RNA-modifying enzymes, but also RNA polymerase subunits, and transcription factors. We show that PfuSmAP1 preferentially binds messenger RNAs and antisense RNAs recognizing a gapped poly(U) sequence with high affinity. Furthermore, we found that SmAP1 co-transcriptionally associates with target RNAs. Our study reveals that in contrast to bacterial Hfq, PfuSmAP1 does not affect the transcriptional activity or the pausing behaviour of archaeal RNA polymerases. We propose that PfuSmAP1 recruits antisense RNAs to target mRNAs and thereby executes its putative regulatory function on the posttranscriptional level.

Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance.

Post-transcriptional modifications have critical roles in tRNA stability and function1-4. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures5,6. Here we identified 2'-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea. Up47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47. The 2'-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for Up47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo. Taken together, our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.

Insights into synthesis and function of KsgA/Dim1-dependent rRNA modifications in archaea.

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.

Expansion segments in bacterial and archaeal 5S ribosomal RNAs.

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.

Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution.

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.

Structural and functional insights into Archaeoglobus fulgidus m2G10 tRNA methyltransferase Trm11 and its Trm112 activator.

tRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N2-methylguanosine (m2G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the Archaeoglobus fulgidus archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin.

A newly identified duplex RNA unwinding activity of archaeal RNase J depends on processive exoribonucleolysis coupled steric occlusion by its structural archaeal loops.

RNase J is a prokaryotic 5'-3' exo/endoribonuclease that functions in mRNA decay and rRNA maturation. Here, we report a novel duplex unwinding activity of mpy-RNase J, an archaeal RNase J from Methanolobus psychrophilus, which enables it to degrade duplex RNAs with hairpins up to 40 bp when linking a 5' single-stranded overhangs of ≥ 7 nt, corresponding to the RNA channel length. A 6-nt RNA-mpy-RNase J-S247A structure reveals the RNA-interacting residues and a steric barrier at the RNA channel entrance comprising two archaeal loops and two helices. Mutagenesis of the residues key to either exoribonucleolysis or RNA translocation diminished the duplex unwinding activity. Substitution of the residues in the steric barrier yielded stalled degradation intermediates at the duplex RNA regions. Thus, an exoribonucleolysis-driven and steric occlusion-based duplex unwinding mechanism was identified. The duplex unwinding activity confers mpy-RNase J the capability of degrading highly structured RNAs, including the bacterial REP RNA, and archaeal mRNAs, rRNAs, tRNAs, SRPs, RNase P and CD-box RNAs, providing an indicative of the potential key roles of mpy-RNase J in pleiotropic RNA metabolisms. Hydrolysis-coupled duplex unwinding activity was also detected in a bacterial RNase J, which may use a shared but slightly different unwinding mechanism from archaeal RNase Js, indicating that duplex unwinding is a common property of the prokaryotic RNase Js.

Role of disordered regions in transferring tyrosine to its cognate tRNA.

Aminoacyl tRNA synthetase (AARS) plays an important role in transferring each amino acid to its cognate tRNA. Specifically, tyrosyl tRNA synthetase (TyrRS) is involved in various functions including protection from DNA damage due to oxidative stress, protein synthesis and cell signaling and can be an attractive target for controlling the pathogens by early inhibition of translation. TyrRS has two disordered regions, which lack a stable 3D structure in solution, and are involved in tRNA synthetase catalysis and stability. One of the disordered regions undergoes disorder-to-order transition (DOT) upon complex formation with tRNA whereas the other remains disordered (DR). In this work, we have explored the importance of these disordered regions using molecular dynamics simulations of both free and RNA-complexed states. We observed that the DOT and DR regions of the first subunit acts as a flap and interact with the acceptor arm of the tRNA. The DOT-DR flap closes when tyrosine (TyrRSTyr) is present at the active site of the complex and opens in the presence of tyrosine monophosphate (TyrRSYMP). The DOT and DR regions of the second subunit interact with the anticodon stem as well as D-loop of the tRNA, which might be involved in stabilizing the complex. The anticodon loop of the tRNA binds to the structured region present in the C-terminal of the protein, which is observed to be flexible during simulations. Detailed energy calculations also show that TyrRSTyr complex has stronger binding energy between tRNA and protein compared to TyrRSYMP; on the contrary, the anticodon is strongly bound in TyrRSYMP. The results obtained in the present study provide additional insights for understanding catalysis and the involvement of disordered regions in Tyr transfer to cognate tRNA.

Archaeosine Modification of Archaeal tRNA: Role in Structural Stabilization.

Archaeosine (G+) is a structurally complex modified nucleoside found quasi-universally in the tRNA of Archaea and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside the Archaea G+ is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G+ at position 15, coupled with its novel molecular structure, led to a hypothesis that G+ stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G+ contributes to tRNA stability and define the biological role of G+, we investigated the consequences of introducing targeted mutations that disrupt the biosynthesis of G+ into the genome of the hyperthermophilic archaeon Thermococcus kodakarensis and the mesophilic archaeon Methanosarcina mazei, resulting in modification of the tRNA with the G+ precursor 7-cyano-7-deazaguansine (preQ0) (deletion of arcS) or no modification at position 15 (deletion of tgtA). Assays of tRNA stability from in vitro-prepared and enzymatically modified tRNA transcripts, as well as tRNA isolated from the T. kodakarensis mutant strains, demonstrate that G+ at position 15 imparts stability to tRNAs that varies depending on the overall modification state of the tRNA and the concentration of magnesium chloride and that when absent results in profound deficiencies in the thermophily of T. kodakarensisIMPORTANCE Archaeosine is ubiquitous in archaeal tRNA, where it is located at position 15. Based on its molecular structure, it was proposed to stabilize tRNA, and we show that loss of archaeosine in Thermococcus kodakarensis results in a strong temperature-sensitive phenotype, while there is no detectable phenotype when it is lost in Methanosarcina mazei Measurements of tRNA stability show that archaeosine stabilizes the tRNA structure but that this effect is much greater when it is present in otherwise unmodified tRNA transcripts than in the context of fully modified tRNA, suggesting that it may be especially important during the early stages of tRNA processing and maturation in thermophiles. Our results demonstrate how small changes in the stability of structural RNAs can be manifested in significant biological-fitness changes.

RNA processing machineries in Archaea: the 5'-3' exoribonuclease aRNase J of the ß-CASP family is engaged specifically with the helicase ASH-Ski2 and the 3'-5' exoribonucleolytic RNA exosome machinery.

A network of RNA helicases, endoribonucleases and exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focussed on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5'-3' exoribonuclease of the ß-CASP family conserved in Euryarchaeota, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, at the vicinity of the ribosome. Proteomic landscapes and direct protein-protein interaction analyses, strengthened by comprehensive phylogenomic studies demonstrated that aRNase J interplay with ASH-Ski2 and a cap exosome subunit. Finally, Thermococcus barophilus whole-cell extract fractionation experiments provide evidences that an aRNase J/ASH-Ski2 complex might exist in vivo and hint at an association of aRNase J with the ribosome that is emphasised in absence of ASH-Ski2. Whilst aRNase J homologues are found among bacteria, the RNA exosome and the Ski2-like RNA helicase have eukaryotic homologues, underlining the mosaic aspect of archaeal RNA machines. Altogether, these results suggest a fundamental role of ß-CASP RNase/helicase complex in archaeal RNA metabolism.

The presence of the ACA box in archaeal H/ACA guide RNAs promotes atypical pseudouridylation.

Archaea and eukaryotes, in addition to protein-only enzymes, also possess ribonucleoproteins containing an H/ACA guide RNA plus four proteins that produce pseudouridine (Ψ). Although typical conditions for these RNA-guided reactions are known, certain variant conditions allow pseudouridylation. We used mutants of the two stem-loops of the Haloferax volcanii sR-h45 RNA that guides three pseudouridylations in 23S rRNA and their target RNAs to characterize modifications under various atypical conditions. The 5' stem-loop produces Ψ2605 and the 3' stem-loop produces Ψ1940 and Ψ1942. The latter two modifications require unpaired "UVUN" (V = A, C, or G) in the target and ACA box in the guide. Ψ1942 modification requires the presence of U1940 (or Ψ1940). Ψ1940 is not produced in the Ψ1942-containing substrate, suggesting a sequential modification of the two residues. The ACA box of a single stem-loop guide is not required when typically unpaired "UN" is up to 17 bases from its position in the guide, but is needed when the distance increases to 19 bases or the N is paired. However, ANA of the H box of the double stem-loop guide is needed even for the 5' typical pseudouridylation. The most 5' unpaired U in a string of U's is converted to Ψ, and in the absence of an unpaired U, a paired U can also be modified. Certain mutants of the Cbf5 protein affect pseudouridylation by the two stem-loops of sR-h45 differently. This study will help elucidate the conditions for production of nonconstitutive Ψ's, determine functions for orphan H/ACA RNAs and in target designing.

In Vivo RNA Chemical Footprinting Analysis in Archaea.

RNA structural conformation and dynamics govern the functional properties of all RNA/RNP. Accordingly, defining changes of RNA structure and dynamics in various conditions may provide detailed insight into how RNA structural properties regulate the function of RNA/RNP. Traditional chemical footprinting analysis using chemical modifiers allows to sample the dynamics and conformation landscape of diverse RNA/RNP. However, many chemical modifiers are limited in their capacity to provide unbiased information reflecting the in vivo RNA/RNP structural landscape. In the recent years, the development of selective-2'-hydroxyl acylation analyzed by primer extension (SHAPE) methodology that uses powerful new chemical modifiers has significantly improved in vitro and in vivo structural probing of secondary and tertiary interactions of diverse RNA species at the single nucleotide level.Although the original discovery of Archaea as an independent domain of life is intimately linked to the technological development of RNA analysis, our understanding of in vivo RNA structural conformation and dynamics in this domain of life remains scarce.This protocol describes the in vivo use of SHAPE chemistry in two evolutionary divergent model Archaea, Sulfolobus acidocaldarius and Haloferax volcanii.

Enzymatic Analysis of Reconstituted Archaeal Exosomes.

The archaeal exosome is a protein complex with phosphorolytic activity. It is built of a catalytically active hexameric ring containing the archaeal Rrp41 and Rrp42 proteins, and a heteromeric RNA-binding platform. The platform contains a heterotrimer of the archaeal Rrp4 and Csl4 proteins (which harbor S1 and KH or Zn-ribbon RNA binding domains), and comprises additional archaea-specific subunits. The latter are represented by the archaeal DnaG protein, which harbors a novel RNA-binding domain and tightly interacts with the majority of the exosome isoforms, and Nop5, known as a part of an rRNA methylating complex and found to associate with the archaeal exosome at late stationary phase. Although in the cell the archaeal exosome exists in different isoforms with heterotrimeric Rrp4-Csl4-caps, in vitro it is possible to reconstitute complexes with defined, homotrimeric caps and to study the impact of each RNA-binding subunit on exoribonucleolytic degradation and on polynucleotidylation of RNA. Here we describe procedures for reconstitution of isoforms of the Sulfolobus solfataricus exosome and for set-up of RNA degradation and polyadenylation assays.

The archaeal RNA chaperone TRAM0076 shapes the transcriptome and optimizes the growth of Methanococcus maripaludis.

TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5' untranslated regions (5'UTRs) of 20-60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5'UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5'UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.

DNA and RNA Stable Isotope Probing of Methylotrophic Methanogenic Archaea.

Methylotrophic methanogenic archaea are an integral part of the carbon cycle in various anaerobic environments. Different from methylotrophic bacteria, methylotrophic methanogens assimilate both, the methyl compound and dissolved inorganic carbon. Here, we present DNA- and RNA-stable isotope probing (SIP) methods involving an effective labeling strategy using 13C-labeled dissolved inorganic carbon (DIC) as carbon source along with methanol as dissimilatory substrate.

Diversity of circular RNAs and RNA ligases in archaeal cells.

Circular RNAs (circRNAs) differ structurally from other types of RNAs and are resistant against exoribonucleases. Although they have been detected in all domains of life, it remains unclear how circularization affects or changes functions of these ubiquitous nucleic acid circles. The biogenesis of circRNAs has been mostly described as a backsplicing event, but in archaea, where RNA splicing is a rare phenomenon, a second pathway for circRNA formation was described in the cases of rRNAs processing, tRNA intron excision, and Box C/D RNAs formation. At least in some archaeal species, circRNAs are formed by a ligation step catalyzed by an atypic homodimeric RNA ligase belonging to Rnl3 family. In this review, we describe archaeal circRNA transcriptomes obtained using high throughput sequencing technologies on Sulfolobus solfataricus, Pyrococcus abyssi and Nanoarchaeum equitans cells. We will discuss the distribution of circular RNAs among the different RNA categories and present the Rnl3 ligase family implicated in the circularization activity. Special focus is given for the description of phylogenetic distributions, protein structures, and substrate specificities of archaeal RNA ligases.

Pyrite formation from FeS and H2S is mediated through microbial redox activity.

The exergonic reaction of FeS with H2S to form FeS2 (pyrite) and H2 was postulated to have operated as an early form of energy metabolism on primordial Earth. Since the Archean, sedimentary pyrite formation has played a major role in the global iron and sulfur cycles, with direct impact on the redox chemistry of the atmosphere. However, the mechanism of sedimentary pyrite formation is still being debated. We present microbial enrichment cultures which grew with FeS, H2S, and CO2 as their sole substrates to produce FeS2 and CH4 Cultures grew over periods of 3 to 8 mo to cell densities of up to 2 to 9 × 106 cells per mL-1 Transformation of FeS with H2S to FeS2 was followed by 57Fe Mössbauer spectroscopy and showed a clear biological temperature profile with maximum activity at 28 °C and decreasing activities toward 4 °C and 60 °C. CH4 was formed concomitantly with FeS2 and exhibited the same temperature dependence. Addition of either penicillin or 2-bromoethanesulfonate inhibited both FeS2 and CH4 production, indicating a coupling of overall pyrite formation to methanogenesis. This hypothesis was supported by a 16S rRNA gene-based phylogenetic analysis, which identified at least one archaeal and five bacterial species. The archaeon was closely related to the hydrogenotrophic methanogen Methanospirillum stamsii, while the bacteria were most closely related to sulfate-reducing Deltaproteobacteria, as well as uncultured Firmicutes and Actinobacteria. Our results show that pyrite formation can be mediated at ambient temperature through a microbially catalyzed redox process, which may serve as a model for a postulated primordial iron-sulfur world.

Indications for a moonlighting function of translation factor aIF5A in the crenarchaeum Sulfolobus solfataricus.

Translation factor a/eIF5A is highly conserved in Eukarya and Archaea. The eukaryal eIF5A protein is required for transit of ribosomes across consecutive proline codons, whereas the function of the archaeal orthologue remains unknown. Here, we provide a first hint for an involvement of Sulfolobus solfataricus (Sso) aIF5A in translation. CRISPR-mediated knock down of the aif5A gene resulted in strong growth retardation, underlining a pivotal function. Moreover, in vitro studies revealed that Sso aIF5A is endowed with endoribonucleolytic activity. Thus, aIF5A appears to be a moonlighting protein that might be involved in protein synthesis as well as in RNA metabolism.

The role of RNA structure in translational regulation by L7Ae protein in archaea.

A recent study has shown that archaeal L7Ae binds to a putative k-turn structure in the 5'-leader of the mRNA of its structural gene to regulate translation. To function as a regulator, the RNA should be unstructured in the absence of protein, but it should adopt a k-turn-containing stem-loop on binding L7Ae. Sequence analysis of UTR sequences indicates that their k-turn elements will be unable to fold in the absence of L7Ae, and we have demonstrated this experimentally in solution using FRET for the Archaeoglobus fulgidus sequence. We have solved the X-ray crystal structure of the complex of the A. fulgidus RNA bound to its cognate L7Ae protein. The RNA adopts a standard k-turn conformation that is specifically recognized by the L7Ae protein, so stabilizing the stem-loop. In-line probing of the natural-sequence UTR shows that the RNA is unstructured in the absence of L7Ae binding, but folds on binding the protein such that the ribosome binding site is occluded. Thus, L7Ae regulates its own translation by switching the conformation of the RNA to alter accessibility.