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Little is known about the variations of nematode mitogenomes (mtDNA). Sequencing a complete mtDNA using a PCR approach remains a challenge due to frequent genome reorganizations and low sequence similarities between divergent nematode lineages. Here, a genome skimming approach based on HiSeq sequencing (shotgun) was used to assemble de novo the first complete mtDNA sequence of a root-knot nematode (Meloidogyne graminicola). An AT-rich genome (84.3%) of 20,030 bp was obtained with a mean sequencing depth superior to 300. Thirty-six genes were identified with a semi-automated approach. A comparison with a gene map of the M. javanica mitochondrial genome indicates that the gene order is conserved within this nematode lineage. However, deep genome rearrangements were observed when comparing with other species of the superfamily Hoplolaimoidea. Repeat elements of 111 bp and 94 bp were found in a long non-coding region of 7.5 kb, as similarly reported in M. javanica and M. hapla. This study points out the power of next generation sequencing to produce complete mitochondrial genomes, even without a reference sequence, and possibly opening new avenues for species/race identification, phylogenetics and population genetics of nematodes., (Copyright © 2014 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

Thiolation of carbon-2 of uridine located in the first position of the anticodons of tRNAUUG(Gln), tRNAUUC(Glu), and tRNAUUU(Lys) is a conserved RNA modification event requiring the 2-thiouridine synthetase Ncs6/Ctu1 in archaea and eukaryotes. Ncs6/Ctu1 activates uridine by adenylation, but its role in sulfur transfer is unclear. Here we show that Mmp1356, the Ncs6/Ctu1 homolog in the archaeon Methanococcus maripaludis, forms a persulfide enzyme adduct with an active site cysteine; this suggests that Mmp1356 directly participates in sulfur transfer as a persulfide carrier. Transposon mutagenesis shows that Mmp1356 is likely to be an essential protein., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The enzyme predominantly used for in vitro run-off RNA synthesis is bacteriophage T7 RNA polymerase. T7 RNA polymerase synthesizes, in addition to run-off products of precise length, transcripts with an additional non-base-paired nucleotide at the 3'-terminus (N+1 product). This contaminating product is extremely difficult to remove. We recently characterized the single-subunit RNA polymerase from marine cyanophage Syn5 and identified its promoter sequence. This marine enzyme catalyses RNA synthesis over a wider range of temperature and salinity than does T7 RNA polymerase. Its processivity is >30,000 nt without significant intermediate products. The requirement for the initiating nucleotide at the promoter is less stringent for Syn5 RNA polymerase as compared to T7 RNA polymerase. A major difference is the precise run-off transcripts with homogeneous 3'-termini synthesized by Syn5 RNA polymerase. Therefore, the enzyme is advantageous for the production of RNAs that require precise 3'-termini, such as tRNAs and RNA fragments that are used for subsequent assembly.

Reconstitution of protein complexes has been a valuable tool to test molecular functions and to interpret in vivo observations. In recent years, a large number of RNA-protein complexes has been identified to regulate gene expression and to be important for a range of cellular functions. In contrast to protein complexes, in vitro analyses of RNA-protein complexes are hampered by the fact that recombinant expression and purification of RNA molecules is more difficult and less well established than for proteins. Here we review the current state of technology available for in vitro experiments with RNAs. We outline the possibilities to produce and purify large amounts of homogenous RNA and to perform the required quality controls. RNA-specific problems such as degradation, 5' and 3' end heterogeneity, co-existence of different folding states, and prerequisites for reconstituting RNAs with recombinantly expressed proteins are discussed. Additionally a number of techniques for the characterization of direct and indirect RNA-protein interactions are explained., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacillus subtilis SC-8 produces an antibiotic that has narrow antagonistic activity against bacteria in the Bacillus cereus group. In B. cereus group bacteria, peptide-activating PlcR (PapR) plays a significant role in regulating the transcription of virulence factors. When B. subtilis SC-8 and B. cereus are co-cultured, PapR is assumed to stimulate antibiotic production by B. subtilis SC-8. To better understand the effect of PapR on this interspecies interaction, the global transcriptome profile of B. subtilis SC-8 was analyzed in the presence of PapR. Significant changes were detected in 12.8 % of the total transcripts. Genes related to amino acid transport and metabolism (16.5 %) and transcription (15 %) were mainly upregulated, whereas genes involved in carbohydrate transport and metabolism (12.7 %) were markedly downregulated. The expression of genes related to transcription, including several transcriptional regulators and proteins involved in tRNA biosynthesis, was increased. The expression levels of genes associated with several transport systems, such as antibiotic, cobalt, and iron complex transporters, was also significantly altered. Among the downregulated genes were transcripts associated with spore formation, the subtilosin A gene cluster, and nitrogen metabolism.

In eukaryotes, transfer RNAs (tRNAs) are transcribed in the nucleus yet function in the cytoplasm; thus, tRNA movement within the cell was believed to be unidirectional--from the nucleus to the cytoplasm. It is now known that mature tRNAs also move in a retrograde direction from the cytoplasm to the nucleus via retrograde tRNA nuclear import, a process that is conserved from yeast to vertebrates. The biological significance of this tRNA nuclear import is not entirely clear. We hypothesized that retrograde tRNA nuclear import might function in proofreading tRNAs to ensure that only proper tRNAs reside in the cytoplasm and interact with the translational machinery. Here we identify two major types of aberrant tRNAs in yeast: a 5', 3' end-extended, spliced tRNA and hypomodified tRNAs. We show that both types of aberrant tRNAs accumulate in mutant cells that are defective in tRNA nuclear traffic, suggesting that they are normally imported into the nucleus and are repaired or degraded. The retrograde pathway functions in parallel with the cytoplasmic rapid tRNA decay pathway previously demonstrated to monitor tRNA quality, and cells are not viable if they lack both pathways. Our data support the hypothesis that the retrograde process provides a newly discovered level of tRNA quality control as a pathway that monitors both end processing of pre-tRNAs and the modification state of mature tRNAs.

tRNA biogenesis in yeast involves the synthesis of the initial transcript by RNA polymerase III followed by processing and controlled degradation in both the nucleus and the cytoplasm. A vast landscape of regulatory elements controlling tRNA stability in yeast has emerged from recent studies. Diverse pathways of tRNA maturation generate multiple stable and unstable intermediates. A significant impact on tRNA stability is exerted by a variety of nucleotide modifications. Pre-tRNAs are targets of exosome-dependent surveillance in the nucleus. Some tRNAs that are hypomodified or bear specific destabilizing mutations are directed to the rapid tRNA decay pathway leading to 5'→3' exonucleolytic degradation by Rat1 and Xrn1. tRNA molecules are selectively marked for degradation by a double CCA at their 3' ends. In addition, under different stress conditions, tRNA half-molecules can be generated by independent endonucleolytic cleavage events. Recent studies reveal unexpected relationships between the subsequent steps of tRNA biosynthesis and the mechanisms controlling its quality and turnover., (Copyright © 2013 John Wiley & Sons, Ltd.)

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459-469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

Streptococcus pneumoniae is a causative agent of nosocomial infections such as pneumonia, meningitis, and septicemia. Penicillin resistance in S. pneumoniae depends in part upon MurM, an aminoacyl-tRNA ligase that attaches L-serine or L-alanine to the stem peptide lysine of Lipid II in cell wall peptidoglycan. To investigate the exact substrates the translation machinery provides MurM, quality control by alanyl-tRNA synthetase (AlaRS) was investigated. AlaRS mischarged serine and glycine to tRNA(Ala), as observed in other bacteria, and also transferred alanine, serine, and glycine to tRNA(Phe). S. pneumoniae tRNA(Phe) has an unusual U4:C69 mismatch in its acceptor stem that prevents editing by phenylalanyl-tRNA synthetase (PheRS), leading to the accumulation of misaminoacylated tRNAs that could serve as substrates for translation or for MurM. Although the peptidoglycan layer of S. pneumoniae tolerates a combination of both branched and linear muropeptides, deletion of MurM results in a reversion to penicillin sensitivity in strains that were previously resistant. However, because MurM is not required for cell viability, the reason for its functional conservation across all strains of S. pneumoniae has remained elusive. We now show that MurM can directly function in translation quality control by acting as a broad specificity lipid-independent trans editing factor that deacylates tRNA. This activity of MurM does not require the presence of its second substrate, Lipid II, and can functionally substitute for the activity of widely conserved editing domain homologues of AlaRS, termed AlaXPs proteins, which are themselves absent from S. pneumoniae.

Glucagon-like peptide-1 (GLP-1) has been shown to protect pancreatic β-cells against cytokine-induced dysfunction and destruction. The mechanisms through which GLP-1 exerts its effects are complex and still poorly understood. The aim of this study was to analyze the protein expression profiles of human islets of Langerhans treated with cytokines (IL-1β and IFN-γ) in the presence or absence of GLP-1 by 2D difference gel electrophoresis and subsequent protein interaction network analysis to understand the molecular pathways involved in GLP-1-mediated β-cell protection. Co-incubation of cytokine-treated human islets with GLP-1 resulted in a marked protection of β-cells against cytokine-induced apoptosis and significantly attenuated cytokine-mediated inhibition of glucose-stimulated insulin secretion. The cytoprotective effects of GLP-1 coincided with substantial alterations in the protein expression profile of cytokine-treated human islets, illustrating a counteracting effect on proteins from different functional classes such as actin cytoskeleton, chaperones, metabolic proteins, and islet regenerating proteins. In summary, GLP-1 alters in an integrated manner protein networks in cytokine-exposed human islets while protecting them against cytokine-mediated cell death and dysfunction. These data illustrate the beneficial effects of GLP-1 on human islets under immune attack, leading to a better understanding of the underlying mechanisms involved, a prerequisite for improving therapies for diabetic patients.

The mitochondrial (mt) genome of the sea anemone Metridium senile contains genes for only two transfer RNAs (tRNAs), tRNAf-Met and tRNATrp. Experiments were conducted to seek evidence for the occurrence of functional tRNAs corresponding to these genes and for the participation of nuclear DNA-encoded tRNAs in mt-protein synthesis. RNA sequences corresponding to the two mt-tRNA genes were located in mitochondria and it was shown that 3'-CC (and possibly A, but no other nucleotide) is added post-transcriptionally to the 3' end of at least 50 % of mt-tRNAf-Met molecules and to a small fraction of the mt-tRNATrp molecules. Using specific oligonucleotide primers based on expected nuclear DNA-encoded tRNAs in a series of RACE experiments, we located the nuclear genes for tRNAGln, tRNAIle, tRNAi-Met, tRNAVal and tRNAThr. Data from Northern blot analyses indicated that mtDNA-encoded tRNAf-Met is limited to mitochondria but that nuclear DNA-encoded tRNAVal and tRNAi-Met are present in the cytoplasm and in mitochondria. These data provide direct evidence that in M. senile, mature, functional tRNAs are transcribed from the mtDNA-encoded tRNAf-Met and tRNATrp genes, and are consistent with the interpretation that both nuclear DNA-encoded tRNAVal and tRNAi-Met are utilized in mitochondrial and cytosolic protein synthesis.

We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9-1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50-88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA(opt)) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.

tRNA synthesis by yeast RNA polymerase III (Pol III) is down-regulated under growth-limiting conditions. This control is mediated by Maf1, a global negative regulator of Pol III transcription. Conserved from yeast to man, Maf1 was originally discovered in Saccharomyces cerevisiae by a genetic approach. Details regarding the molecular basis of Pol III repression by Maf1 are now emerging from the recently reported structural and biochemical data on Pol III and Maf1. The phosphorylation status of Maf1 determines its nuclear localization and interaction with the Pol III complex and several Maf1 kinases have been identified to be involved in Pol III control. Moreover, Maf1 indirectly affects tRNA maturation and decay. Here I discuss the current understanding of the mechanisms that oversee the Maf1-mediated regulation of Pol III activity and the role of Maf1 in the control of tRNA biosynthesis in yeast. This article is part of a Special Issue entitled: Transcription by Odd Pols., (Copyright © 2012 Elsevier B.V. All rights reserved.)

tRNA genes (tDNAs) have been known to have barrier insulator function in budding yeast, Saccharomyces cerevisiae, for over a decade. tDNAs also play a role in genome organization by clustering at sites in the nucleus and both of these functions are dependent on the transcription factor TFIIIC. More recently TFIIIC bound sites devoid of pol III, termed Extra-TFIIIC sites (ETC) have been identified in budding yeast and these sites also function as insulators and affect genome organization. Subsequent studies in Schizosaccharomyces pombe showed that TFIIIC bound sites were insulators and also functioned as Chromosome Organization Clamps (COC); tethering the sites to the nuclear periphery. Very recently studies have moved to mammalian systems where pol III genes and their associated factors have been investigated in both mouse and human cells. Short interspersed nuclear elements (SINEs) that bind TFIIIC, function as insulator elements and tDNAs can also function as both enhancer - blocking and barrier insulators in these organisms. It was also recently shown that tDNAs cluster with other tDNAs and with ETCs but not with pol II transcribed genes. Intriguingly, TFIIIC is often found near pol II transcription start sites and it remains unclear what the consequences of TFIIIC based genomic organization are and what influence pol III factors have on pol II transcribed genes and vice versa. In this review we provide a comprehensive overview of the known data on pol III factors in insulation and genome organization and identify the many open questions that require further investigation. This article is part of a Special Issue entitled: Transcription by Odd Pols., (Copyright © 2012 Elsevier B.V. All rights reserved.)

How eukaryotic RNA polymerases switch from elongation to termination is unknown. Pol III subunits Rpc53 and Rpc37 (C53/37) form a heterodimer homologous to TFIIFβ/α. C53/37 promotes efficient termination and together with C11 also mediates pol III recycling in vitro. We previously developed Schizosaccharomyces pombe strains that report on two pol III termination activities: RNA oligo(U) 3'-end cleavage, and terminator readthrough. We randomly mutagenized C53 and C37 and isolated many C37 mutants with terminator readthrough but no comparable C53 mutants. The majority of C37 mutants have strong phenotypes with up to 40% readthrough and map to a C-terminal tract previously localized near Rpc2p in the pol III active center while a minority represent a distinct class with weaker phenotype, less readthrough and 3'-oligo(U) lengthening. Nascent pre-tRNAs released from a terminator by C37 mutants have shorter 3'-oligo(U) tracts than in cleavage-deficient C11 double mutants indicating RNA 3'-end cleavage during termination. We asked whether termination deficiency affects transcription output in the mutants in vivo both by monitoring intron-containing nascent transcript levels and (14)C-uridine incorporation. Surprisingly, multiple termination mutants have no decrease in transcript output relative to controls. These data are discussed in context of current models of pol III transcription.

Background: Protein arginine methylation is a post-translational modification involved in important biological processes such as transcription and RNA processing. This modification is catalyzed by both type I and II protein arginine methyltransferases (PRMTs). One of the most conserved type I PRMTs is PRMT1, the homolog of which is Hmt1 in Saccharomyces cerevisiae. Hmt1 has been shown to play a role in various gene expression steps, such as promoting the dynamics of messenger ribonucleoprotein particle (mRNP) biogenesis, pre-mRNA splicing, and silencing of chromatin. To determine the full extent of Hmt1's involvement during gene expression, we carried out a genome-wide location analysis for Hmt1., Results: A comprehensive genome-wide binding profile for Hmt1 was obtained by ChIP-chip using NimbleGen high-resolution tiling microarrays. Of the approximately 1000 Hmt1-binding sites found, the majority fall within or proximal to an ORF. Different occupancy patterns of Hmt1 across genes with different transcriptional rates were found. Interestingly, Hmt1 occupancy is found at a number of other genomic features such as tRNA and snoRNA genes, thereby implicating a regulatory role in the biogenesis of these non-coding RNAs. RNA hybridization analysis shows that Hmt1 loss-of-function mutants display higher steady-state tRNA abundance relative to the wild-type. Co-immunoprecipitation studies demonstrate that Hmt1 interacts with the TFIIIB component Bdp1, suggesting a mechanism for Hmt1 in modulating RNA Pol III transcription to regulate tRNA production., Conclusions: The genome-wide binding profile of Hmt1 reveals multiple potential new roles for Hmt1 in the control of eukaryotic gene expression, especially in the realm of non-coding RNAs. The data obtained here will provide an important blueprint for future mechanistic studies on the described occupancy relationship for genomic features bound by Hmt1.

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.

Mycobacterium tuberculosis serine/threonine protein kinases (STPKs) are responsible for orchestrating critical metabolic and physiological changes that dictate mycobacterial growth adaptation. Previously, we established that PknK participates in regulatory pathways that slow the growth of M. tuberculosis in a variety of in vitro stress environments and during persistent infection in mice. In the present study, we have elaborated on the mechanism of PknK-mediated regulation. Through transcription profiling of wild-type H37Rv and a ΔpknK mutant strain during logarithmic and stationary growth phases, we determined that PknK regulates the expression of a large subset of tRNA genes so that regulation is synchronized with growth phase and cellular energy status. Elevated levels of wild-type M. tuberculosis PknK (PknK(Mtb)), but not phosphorylation-defective PknK(Mtb), in Mycobacterium smegmatis cause significant retardation of the growth rate and altered colony morphology. We investigated a role for PknK in translational control and established that PknK directs the inhibition of in vitro transcription and translation processes in a phosphorylation-dependent manner. Increasing concentrations of ATP or PknK exert cooperative effects and enhance the inhibitory function of PknK. Furthermore, truncation and mutational analyses of PknK revealed that PknK is autoregulated via intramolecular interactions with its C-terminal region. Significantly, the invariant lysine 55 residue was only essential for activity in the full-length PknK protein, and the truncated mutant proteins were active. A model for PknK autoregulation is proposed and discussed.

Abstract The biogenesis of most eukaryotic kinds of RNA requires nuclear export, which is mediated by a variety of specific nuclear transport receptors. The nuclear export receptors Exportin-t (Exp-t) and Exportin 5 (Exp5), and their homologues, are involved in the export of transfer RNA to the cytoplasm. Exp5 is further involved in additional nucleocytoplasmic transport pathways, which include nuclear export of microRNA precursors (pre-miRNAs) and pre-60S ribosomal subunits. Inactivation of Exp5 results in nuclear accumulation of pre-miRNAs and perturbation of gene expression, and its mutation was recently found in malignant diseases. Here, we compare the cellular function of Exp5 and Exp-t with focus on Exp5 substrates and its role in diseases.

Target of rapamycin (TOR)-dependent signaling and the control of cell growth is deregulated in many cancers. However, the signaling molecules downstream of TOR that coordinately regulate the synthesis of ribosomes and tRNAs are not well defined. Here, we show in yeast that conserved kinases of the LAMMER/Cdc-like and GSK-3 families function downstream of TOR complex 1 to repress ribosome and tRNA synthesis in response to nutrient limitation and other types of cellular stress. As a part of this response, we found that the LAMMER kinase Kns1 is differentially expressed and hyperphosphorylated and accumulates in the nucleus after rapamycin treatment, whereupon it primes the phosphorylation of the RNA polymerase III subunit Rpc53 by a specific GSK-3 family member, Mck1. In cooperation with another polymerase subunit, Rpc11, this phosphorylation of Rpc53 modifies the function of the enzyme and together with dephosphorylation of the Maf1 repressor inhibits the growth-promoting activity of RNA polymerase III transcription., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Posttranscriptional modifications of transfer RNAs (tRNAs) are critical for all core aspects of tRNA function, such as folding, stability, and decoding. Most tRNA modifications were discovered in the 1970s; however, the near-complete description of the genes required to introduce the full set of modifications in both yeast and Escherichia coli is very recent. This led to a new appreciation of the key roles of tRNA modifications and tRNA modification enzymes as checkpoints for tRNA integrity and for integrating translation with other cellular functions such as transcription, primary metabolism, and stress resistance. A global survey of tRNA modification enzymes shows that the functional constraints that drive the presence of modifications are often conserved, but the solutions used to fulfill these constraints differ among different kingdoms, organisms, and species.

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.

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. In vitro transcription or chemical synthesis are the principal methods to produce RNA. Here, we describe an alternative method allowing RNA production in bacteria and its purification by liquid chromatography. In a few days, between 10 and 100 mg of pure RNA are obtained with this technique.

Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg(2+) and K(+) and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS(C17S)) in apo form (without ATP bound). EcSPS(C17S) crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.

Cysteine (Cys) has unique chemical properties of catalysis, metal chelation, and protein stabilization. While Cys biosynthesis is assumed to be very ancient, the actual time of origin of these metabolic pathways remains unknown. Here, we use the molecular clocks of protein folds and fold superfamilies to time the origin of Cys biosynthesis. We find that the tRNA-dependent biosynthetic pathway appeared ~3.5 billion years ago while the tRNA-independent counterpart emerged ~500 million years later. A deep analysis of the origins of Cys biosynthesis in the context of emerging biochemistry uncovers some intriguing features of the planetary environment of early Earth. Results suggest that iron-sulfur (Fe-S) proteins that use cysteinyl sulfur to bind iron atoms were not the first to arise in evolution. Instead, their origin coincides with the appearance of the first Cys biosynthetic pathway. It is therefore likely that Cys did not play an important role in the make up of primordial protein molecules and that Fe-S clusters were not part of active sites at the beginning of biological history.

Maf1 is negative regulator of RNA polymerase III in yeast. We observed high levels of both primary transcript and end-matured, intron-containing pre-tRNAs in the maf1Δ strain. This pre-tRNA accumulation could be overcome by transcription inhibition, arguing against a direct role of Maf1 in tRNA maturation and suggesting saturation of processing machinery by the increased amounts of primary transcripts. Saturation of the tRNA exportin, Los1, is one reason why end-matured intron-containing pre-tRNAs accumulate in maf1Δ cells. However, it is likely possible that other components of the processing pathway are also limiting when tRNA transcription is increased. According to our model, Maf1-mediated transcription control and nuclear export by Los1 are two major stages of tRNA biosynthesis that are regulated by environmental conditions in a coordinated manner.

TORC1 is a conserved multisubunit kinase complex that regulates many aspects of eukaryotic growth including the biosynthesis of ribosomes. The TOR protein kinase resident in TORC1 is responsive to environmental cues and is potently inhibited by the natural product rapamycin. Recent characterization of the rapamycin-sensitive phosphoproteome in yeast has yielded insights into how TORC1 regulates growth. Here, we show that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (Ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of the transcriptional repressors Stb3, Dot6 and Tod6. Deletion of STB3, DOT6 and TOD6 partially bypasses the growth and cell size defects of an sch9 strain and reveals interdependent regulation of both Ribi and RP gene expression, and other aspects of Ribi. Dephosphorylation of Stb3, Dot6 and Tod6 enables recruitment of the RPD3L histone deacetylase complex to repress Ribi/RP gene promoters. Taken together with previous studies, these results suggest that Sch9 is a master regulator of ribosome biogenesis through the control of Ribi, RP, ribosomal RNA and tRNA gene transcription.

Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn(2+). The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn(2+) per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn(2+)-coordinating cysteines in TbADAT2 yields a functional enzyme with a single-bound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn(2+). These observations raise the possibility of an unusual Zn(2+) coordination interface with important implications for the function and evolution of editing deaminases.

Cellular total RNA level is usually stable, although it may increase gradually during growth or seed germination, or decrease gradually under environmental stresses. However, we found that plant cell RNA could be doubled within 48 h in response to herbicide-induced Mg-protoporphyrin and heme accumulation or a high level of sugar treatment. This rapid RNA multiplication is important for effective cellular resistance to oxidative stress, such as high-light and herbicide co-stress conditions, where the plastid-signalling defective mutant gun1 shows an apparent phenotype (more severe photobleaching). Hexokinase is required for sugar-induced RNA multiplication. While both sugar and Mg-protoporphyrin IX require plastid protein GUN1 and a nuclear transcription factor ABI4, haem appears to function through an independent pathway to control RNA multiplication. The transcription co-factor CAAT binding protein mediates the rapid RNA multiplication in plant cells in all the cases., (© 2011 Blackwell Publishing Ltd.)

tRNA modifications are important for decoding, translation accuracy, and structural integrity of tRNAs. Archaeal tRNAs contain at least 47 different tRNA modifications, some of them, including archaeosine, agmatidine, and mimG, are specific to the archaeal domain. The biosynthetic pathways for these complex signature modifications have recently been elucidated and are extensively described in this review. Archaeal organisms still lag Escherichia coli and Saccharomyces cerevisiae in terms of genetic characterization and in vivo function of tRNA modifications. However, recent advances in the model Haloferax volcanii, described here, should allow closing this gap soon. Consequently, an update on experimental characterizations of archaeal tRNA modification genes and proteins is given to set the stage for future work in this field., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Maf1 protein is a global negative regulator of RNA polymerase (Pol) III transcription conserved from yeast to man. We report that phosphorylation of Maf1 by casein kinase II (CK2), a highly evolutionarily conserved eukaryotic kinase, is required for efficient Pol III transcription. Both recombinant human and yeast CK2 were able to phosphorylate purified human or yeast Maf1, indicating that Maf1 can be a direct substrate of CK2. Upon transfer of Saccharomyces cerevisiae from repressive to favorable growth conditions, CK2 activity is required for the release of Maf1 from Pol III bound to a tRNA gene and for subsequent activation of tRNA transcription. In a yeast strain lacking Maf1, CK2 inhibition showed no effect on tRNA synthesis, confirming that CK2 activates Pol III via Maf1. Additionally, CK2 was found to associate with tRNA genes, and this association is enhanced in absence of Maf1, especially under repressive conditions. These results corroborate the previously reported TFIIIB-CK2 interaction and indicate an important role of CK2-mediated Maf1 phosphorylation in triggering Pol III activation.

Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.

Purine nucleotides are structural components of the genetic material, function as phosphate donors, participate in cellular signaling, are cofactors in enzymatic reactions, and constitute the main carriers of cellular energy. Thus, imbalances in A/G nucleotide biosynthesis affect nearly the whole cellular metabolism and must be tightly regulated. We have identified a substitution mutation (G388D) that reduces the activity of the GMP synthase Gua1 in budding yeast and the total G-nucleotide pool, leading to precipitous reductions in the GDP/GTP ratio and ATP level in vivo. gua1-G388D strongly reduces the rate of growth, impairs general protein synthesis, and derepresses translation of GCN4 mRNA, encoding a transcriptional activator of diverse amino acid biosynthetic enzymes. Although processing of pre-tRNA(i)(Met) and other tRNA precursors, and the aminoacylation of tRNA(i)(Met) are also strongly impaired in gua1-G388D cells, tRNA(i)(Met)-containing complexes with the macromolecular composition of the eIF2·tRNA(i)(Met.)GTP complex (TC) and the multifactor complex (MFC) required for translation initiation accumulate ∼10-fold in gua1-G388D cells and, to a lesser extent, in wild-type (WT) cells treated with 6-azauracil (6AU). Consistently, addition of an external supply of guanine reverts all the phenotypes of gua1-G388D cells, but not those of gua1-G388D Δhpt1 mutants unable to refill the internal GMP pool through the salvage pathway. These and other findings suggest that a defect in guanine nucleotide biosynthesis evokes a reduction in the rate of general protein synthesis by impairing multiple steps of the process, disrupts the gene-specific reinitiation mechanism for translation of GCN4 mRNA and has far-reaching effects in cell biology and metabolism.

tRNA biology has come of age, revealing an unprecedented level of understanding and many unexpected discoveries along the way. This review highlights new findings on the diverse pathways of tRNA maturation, and on the formation and function of a number of modifications. Topics of special focus include the regulation of tRNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions, emerging evidence of signaling pathways involving tRNA and cleavage fragments, and the sophisticated intracellular tRNA trafficking that occurs during and after biosynthesis.

The c-Myc oncoprotein promotes cell growth by enhancing ribosomal biogenesis. Overexpression of c-Myc and aberrant ribosomal biogenesis lead to deregulated cell growth and tumorigenesis. Hence, c-Myc activity and ribosomal biogenesis must be tightly coordinated during normal homeostasis. We previously found that ribosomal protein L11 inhibits c-Myc activity by blocking the recruitment of its co-activator transformation/transcription domain-associated protein (TRRAP) to the promoter regions of c-Myc target genes that are transcribed by RNA polymerases I and II. In this study, we extended the role of L11 to the regulation of c-Myc-driven transcription of the 5 S rRNA and tRNA genes by RNA polymerase III. L11 co-resided with c-Myc at the 5 S rRNA and tRNA genes and significantly inhibited the binding of TRRAP to these genes. Knocking down endogenous L11 enhanced c-Myc-dependent transcription of these genes. Interestingly, in response to ribosomal stress induced by the treatment of cells with a low dose of actinomycin D or serum starvation, L11 binding to these genes was increased, and inversely TRRAP binding to these genes was decreased. Consistently, knockdown of L11 rescued the reduction of the expression of these genes by the two treatments. These results demonstrate that L11 suppresses c-Myc-dependent and RNA polymerase III-catalyzed transcription of 5 S rRNA and tRNA genes in response to ribosomal stress, ensuring a tight coordination between c-Myc activity and ribosomal biogenesis.

Dr1 (also known as NC2beta) was identified as a repressor of RNA polymerase (pol) II transcription. It was subsequently shown to inhibit pol III transcription when expressed at high levels in vitro or in yeast cells. However, endogenous Dr1 was not detected at pol III-transcribed genes in growing yeast. In contrast, we demonstrate that endogenous Dr1 is present at pol III templates in human cells, as is its dimerization partner DRAP1 (also called NC2alpha). Expression of tRNA by pol III is selectively enhanced by RNAi-mediated depletion of endogenous human Dr1, but we found no evidence that DRAP1 influences pol III output in vivo. A stable association was detected between endogenous Dr1 and the pol III-specific transcription factor Brf1. This interaction may recruit Dr1 to pol III templates in vivo, as crosslinking to these sites increases following Brf1 induction. On the basis of these data, we conclude that the physiological functions of human Dr1 include regulation of pol III transcription.

The chloroplast genome of a dinoflagellate consists of a group of small circular DNA molecules (minicircles), most of which carry a single gene. With RT-PCR, primer extension, and Northern analyses, we show that the entire minicircle is transcribed and that some minicircles can produce RNAs larger than themselves. Using an RNA ligase-mediated rapid amplification of cDNA ends method, we were able to detect large processed precursors that are generated by endonucleolytic cleavage of an even longer molecule. This cleavage produces the mature mRNA 3'-end and at the same time the 5'-end of the precursor. The tRNAs encoded by the petD and psbE minicircles appear to be processed in the same way. We propose a "rolling circle" model for chloroplast transcription in which transcription would proceed continuously around the minicircular DNA to produce transcripts larger than the minicircle itself. These transcripts would be further processed into discrete mature mRNAs and tRNAs.

We have found earlier that Tor1 binds to 5S rDNA chromatin but the functional significance has not been established. Here, we show that association with 5S rDNA chromatin is necessary for TOR complex 1 (TORC1) to regulate the synthesis of 5S ribosomal RNA and transfer RNAs (tRNAs) by RNA polymerase (Pol) III, as well as the phosphorylation and binding to Pol III-transcribed genes of the Pol III repressor Maf1. Interestingly, TORC1 does not bind to tRNA genes, suggesting that TORC1 modulates tRNA synthesis indirectly through Maf1 phosphorylation at the rDNA loci. We also find that Maf1 cytoplasmic localization is dependent on the SSD1-v allele. In W303 cells that carry the SSD1-d allele, Maf1 is constitutively nuclear but its nucleolar localization is inhibited by TORC1, indicating that TORC1 regulates nucleoplasm-to-nucleolus transport of Maf1. Finally, we show that TORC1 interacts with Maf1 in vivo and phosphorylates Maf1 in vitro, and regulates Maf1 nucleoplasm-to-nucleolus translocation. Together, these observations provide new insights into the chromatin-dependent mechanism by which TORC1 controls transcription by Pol III.

Stress-induced phosphorylation of eIF2alpha inhibits global protein synthesis to conserve energy for repair of stress-induced damage. Stress-induced translational arrest is observed in cells expressing a nonphosphorylatable eIF2alpha mutant (S51A), which indicates the existence of an alternative pathway of translational control. In this paper, we show that arsenite, heat shock, or ultraviolet irradiation promotes transfer RNA (tRNA) cleavage and accumulation of tRNA-derived, stress-induced small RNAs (tiRNAs). We show that angiogenin, a secreted ribonuclease, is required for stress-induced production of tiRNAs. Knockdown of angiogenin, but not related ribonucleases, inhibits arsenite-induced tiRNA production and translational arrest. In contrast, knockdown of the angiogenin inhibitor RNH1 enhances tiRNA production and promotes arsenite-induced translational arrest. Moreover, recombinant angiogenin, but not RNase 4 or RNase A, induces tiRNA production and inhibits protein synthesis in the absence of exogenous stress. Finally, transfection of angiogenin-induced tiRNAs promotes phospho-eIF2alpha-independent translational arrest. Our results introduce angiogenin and tiRNAs as components of a phospho-eIF2alpha-independent stress response program.

Transfer-RNA (tRNA) molecules are essential players in protein biosynthesis. They are transcribed as precursors, which have to be extensively processed at both ends to become functional adaptors in protein synthesis. Two endonucleases that directly interact with the tRNA moiety, RNase P and tRNase Z, remove extraneous nucleotides on the molecule's 5'- and 3'-side, respectively. The ribonucleoprotein enzyme RNase P was identified almost 40 years ago and is considered a vestige from the "RNA world". Here, we present the state of affairs on prokaryotic RNase P, with a focus on recent findings on its role in RNA metabolism. tRNase Z was only identified 6 years ago, and we do not yet have a comprehensive understanding of its function. The current knowledge on prokaryotic tRNase Z in tRNA 3'-processing is reviewed here. A second, tRNase Z-independent pathway of tRNA 3'-end maturation involving 3'-exonucleases will also be discussed.

Queuosine (Q) and archaeosine (G(+)) are hypermodified ribonucleosides found in tRNA. Q is present in the anticodon region of tRNA(GUN) in Eukarya and Bacteria, while G(+) is found at position 15 in the D-loop of archaeal tRNA. Prokaryotes produce these 7-deazaguanosine derivatives de novo from GTP through the 7-cyano-7-deazaguanine (pre-Q(0)) intermediate, but mammals import the free base, queuine, obtained from the diet or the intestinal flora. By combining the results of comparative genomic analysis with those of genetic studies, we show that the first enzyme of the folate pathway, GTP cyclohydrolase I (GCYH-I), encoded in Escherichia coli by folE, is also the first enzyme of pre-Q(0) biosynthesis in both prokaryotic kingdoms. Indeed, tRNA extracted from an E. coli DeltafolE strain is devoid of Q and the deficiency is complemented by expressing GCYH-I-encoding genes from different bacterial or archaeal origins. In a similar fashion, tRNA extracted from a Haloferax volcanii strain carrying a deletion of the GCYH-I-encoding gene contains only traces of G(+). These results link the production of a tRNA-modified base to primary metabolism and further clarify the biosynthetic pathway for these complex modified nucleosides.

Background: Up to now, there have been three published versions of a yeast genome-scale metabolic model: iFF708, iND750 and iLL672. All three models, however, lack a detailed description of lipid metabolism and thus are unable to be used as integrated scaffolds for gaining insights into lipid metabolism from multilevel omic measurement technologies (e.g. genome-wide mRNA levels). To overcome this limitation, we reconstructed a new version of the Saccharomyces cerevisiae genome-scale model, iIN800 that includes a more rigorous and detailed description of lipid metabolism., Results: The reconstructed metabolic model comprises 1446 reactions and 1013 metabolites. Beyond incorporating new reactions involved in lipid metabolism, we also present new biomass equations that improve the predictive power of flux balance analysis simulations. Predictions of both growth capability and large scale in silico single gene deletions by iIN800 were consistent with experimental data. In addition, 13C-labeling experiments validated the new biomass equations and calculated intracellular fluxes. To demonstrate the applicability of iIN800, we show that the model can be used as a scaffold to reveal the regulatory importance of lipid metabolism precursors and intermediates that would have been missed in previous models from transcriptome datasets., Conclusion: Performing integrated analyses using iIN800 as a network scaffold is shown to be a valuable tool for elucidating the behavior of complex metabolic networks, particularly for identifying regulatory targets in lipid metabolism that can be used for industrial applications or for understanding lipid disease states.

RNA polymerase (pol) III transcription, responsible for the synthesis of various stable RNAs, including 5 S rRNAs and tRNAs, is regulated by oncogenic proteins and tumor suppressors. Although it is well established that RNA pol III-dependent transcription is deregulated in transformed cells and malignant tumors, it has not been determined whether this represents a cause or consequence of these processes. We show that Rat1a fibroblasts undergoing oncogenic transformation by the TATA-binding protein or c-Myc display enhanced RNA pol III transcription. Decreased expression of the RNA pol III-specific transcription factor Brf1 prevented this increase in RNA pol III transcription. Although the overall proliferation rates of these cells remained unchanged, the ability of cells to grow in an anchorage-independent manner and form tumors in mice was markedly reduced. Although overexpression of Brf1 modestly stimulated RNA pol III transcription, expression of a phosphomimic, Brf1-T145D, more significantly induced transcription. However, these increases in transcription were not sufficient to promote cellular transformation. Together, these results demonstrate that enhanced RNA pol III transcription is essential for anchorage-independent growth and tumorigenesis and that these events can be uncoupled from effects on anchorage-dependent proliferation.

Transcriptional repression of ribosomal components and tRNAs is coordinately regulated in response to a wide variety of environmental stresses. Part of this response involves the convergence of different nutritional and stress signaling pathways on Maf1, a protein that is essential for repressing transcription by RNA polymerase (pol) III in Saccharomyces cerevisiae. Here we identify the functions buffering yeast cells that are unable to down-regulate transcription by RNA pol III. MAF1 genetic interactions identified in screens of non-essential gene-deletions and conditionally expressed essential genes reveal a highly interconnected network of 64 genes involved in ribosome biogenesis, RNA pol II transcription, tRNA modification, ubiquitin-dependent proteolysis and other processes. A survey of non-essential MAF1 synthetic sick/lethal (SSL) genes identified six gene-deletions that are defective in transcriptional repression of ribosomal protein (RP) genes following rapamycin treatment. This subset of MAF1 SSL genes included MED20 which encodes a head module subunit of the RNA pol II Mediator complex. Genetic interactions between MAF1 and subunits in each structural module of Mediator were investigated to examine the functional relationship between these transcriptional regulators. Gene expression profiling identified a prominent and highly selective role for Med20 in the repression of RP gene transcription under multiple conditions. In addition, attenuated repression of RP genes by rapamycin was observed in a strain deleted for the Mediator tail module subunit Med16. The data suggest that Mediator and Maf1 function in parallel pathways to negatively regulate RP mRNA and tRNA synthesis., Competing Interests: The authors have declared that no competing interests exist.

MnmC catalyses the last two steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA. Previously, we reported that this bifunctional enzyme is encoded by the yfcK open reading frame in the Escherichia coli K12 genome. However, the mechanism of its activity, in particular the potential structural and functional dependence of the domains responsible for catalyzing the two modification reactions, remains unknown. With the aid of the protein fold-recognition method, we constructed a structural model of MnmC in complex with the ligands and target nucleosides and studied the role of individual amino acids and entire domains by site-directed and deletion mutagenesis, respectively. We found out that the N-terminal domain contains residues responsible for binding of the S-adenosylmethionine cofactor and catalyzing the methylation of nm(5)s(2)U to form mnm(5)s(2)U, while the C-terminal domain contains residues responsible for binding of the FAD cofactor. Further, point mutants with compromised activity of either domain can complement each other to restore a fully functional enzyme. Thus, in the conserved fusion protein MnmC, the individual domains retain independence as enzymes. Interestingly, the N-terminal domain is capable of independent folding, while the isolated C-terminal domain is incapable of folding on its own, a situation similar to the one reported recently for the rRNA modification enzyme RsmC., ((c) 2008 Wiley-Liss, Inc.)

The vaccinia virus (VV) replicates robustly and alters the progression of the cell cycle via an unknown mechanism. Herein, we provide evidence for the existence of a unique VV infection-induced cell cycle control mechanism. The regulation is correlated with the inactivation of p53 and Rb, which are associated with the RNA polymerase III transcription factor B (TFIIIB) subunits, TBP and Brf1 respectively. VV infection induced the expression of Mdm2 and its translocation into the nucleus, thereby resulting in a disruption of p53. VV also stimulated the expression of TFIIIB and TFIIIC, and consequently induced tRNA synthesis. On the other hand, the total level of Rb was not significantly influenced, but the level of hypo-phosphorylated Rb was enhanced, partially due to the VV-induced downregulation of cyclin-dependent kinases 4 and 6. However, the hypo-phosphorylated Rb appeared to be largely sequestered into a complex with Brf1, which resulted in the blockage of Rb function to repress E2F1 transactivation, thereby leading to a moderately higher proportion of cells in the S and G(2) phases. Conversely, the enforced expression of exogenous Rb restored the normally observed cell cycle patterns. Overall, these controls may contribute to the efficient replication of the virus in rapidly growing cells.

TRNAs from all organisms contain posttranscriptionally modified nucleosides, which are derived from the four canonical nucleosides. In most tRNAs that read codons beginning with U, adenosine in the position 37 adjacent to the 3' position of the anticodon is modified to N(6)-(Delta(2)-isopentenyl) adenosine (i(6)A). In many bacteria, such as Escherichia coli, this residue is typically hypermodified to N(6)-isopentenyl-2-thiomethyladenosine (ms(2)i(6)A). In a few bacteria, such as Salmonella typhimurium, ms(2)i(6)A can be further hydroxylated to N(6)-(cis-4-hydroxyisopentenyl)-2-thiomethyladenosine (ms(2)io(6)A). Although the enzymes that introduce the respective modifications (prenyltransferase MiaA, methylthiotransferase MiaB, and hydroxylase MiaE) have been identified, their structures remain unknown and sequence-function relationships remain obscure. We carried out sequence analysis and structure prediction of MiaA, MiaB, and MiaE, using the protein fold-recognition approach. Three-dimensional models of all three proteins were then built using a new modeling protocol designed to overcome uncertainties in the alignments and divergence between the templates. For MiaA and MiaB, the catalytic core was built based on the templates from the P-loop NTPase and Radical-SAM superfamilies, respectively. For MiaB, we have also modeled the C-terminal TRAM domain and the newly predicted N-terminal flavodoxin-fold domain. For MiaE, we confidently predict that it shares the three-dimensional fold with the ferritin-like four-helix bundle proteins and that it has a similar active site and mechanism of action to diiron carboxylate enzymes, in particular, methane monooxygenase (E.C.1.14.13.25) that catalyses the biological hydroxylation of alkanes. Our models provide the first structural platform for enzymes involved in the biosynthesis of i(6)A, ms(2)i(6)A, and ms(2)io(6)A, explain the data available from the literature and will help to design further experiments and interpret their results., ((c) 2007 Wiley-Liss, Inc.)

Several significant structures of transfer ribonucleic acid (tRNA) maturation enzymes complexed with precursor tRNA or fragments thereof have been published recently, providing detailed knowledge of enzyme-tRNA recognition and catalytic strategies. In addition to reinforcing the general principles of RNA-protein interaction, the new structures highlight both the features of composite RNA recognition by multiple enzyme subunits and the pronounced RNA structural flexibility in or near the active site in all cases. These structural principles provide plausible explanations for the exquisite specificity and catalytic power of these enzymes and, in the case of evolutionary adaptation, for the ability of some enzymes to develop novel specificities.