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Hadal trenches represent the deepest part of the ocean and are dynamic depocenters with intensified prokaryotic activity. Here, we explored the distribution and drivers of prokaryotic and viral abundance from the ocean surface and 40 cm into sediments in two hadal trench regions with contrasting surface productivity. In the water column, prokaryotic and viral abundance decreased with water depth before reaching a rather stable level at similar to 4000 m depth at both trench systems, while virus to prokaryote ratios were increasing with depth, presumably reflecting the declining availability of organic material. Prokaryotic and viral abundances in sediments were lower at the adjacent abyssal sites than at the hadal sites and declined exponentially with sediment depth, closely tracking the attenuation of total organic carbon (TOC) content. In contrast, hadal sediment exhibited erratic depth profiles of prokaryotes and viruses with many subsurface peaks. The prokaryotic abundance correlated well to extensive fluctuations in TOC content at centimeter scale, which were likely caused by recurring mass wasting events. Yet while prokaryotic and viral abundances cross correlated well in the abyssal sediments, there was no clear correlation in the hadal sites. The results suggested that dynamic depositional conditions and higher substrate availability result in a high spatial heterogeneity in viral and prokaryotic abundances in hadal sediments in comparison to more stable abyssal settings. We argue that these conditions enhance the relatively importance of viruses for prokaryotic mortality and carbon recycling in hadal settings.

Polymyxins are increasingly used as the critical last-resort therapeutic options for multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance has increased gradually over the past few years. Although studies on polymyxin mechanisms are expanding, systemwide analyses of the underlying mechanism for polymyxin resistance and stress response are still lacking. To understand how Klebsiella pneumoniae adapts to colistin (polymyxin E) pressure, we carried out proteomic analysis of a K. pneumoniae strain cultured with different concentrations of colistin. Our results showed that the proteomic responses to colistin treatment in K. pneumoniae involve several pathways, including (i) gluconeogenesis and the tricarboxylic acid (TCA) cycle, (ii) arginine biosynthesis, (iii) porphyrin and chlorophyll metabolism, and (iv) enterobactin biosynthesis. Interestingly, decreased abundances of class A β-lactamases, including TEM, SHV-11, and SHV-4, were observed in cells treated with colistin. Moreover, we present comprehensive proteome atlases of paired polymyxin-susceptible and -resistant K. pneumoniae strains. The polymyxin-resistant strain Ci, a mutant of K. pneumoniae ATCC BAA 2146, showed a missense mutation in crrB. This crrB mutant, which displayed lipid A modification with 4-amino-4-deoxy-L-arabinose (L-Ara4N) and palmitoylation, showed striking increases in the expression of CrrAB, PmrAB, PhoPQ, ArnBCADT, and PagP. We hypothesize that crrB mutations induce elevated expression of the arnBCADTEF operon and pagP via PmrAB and PhoPQ. Moreover, the multidrug efflux pump KexD, which was induced by crrB mutation, also contributed to colistin resistance. Overall, our results demonstrated proteomic responses to colistin treatment and the mechanism of CrrB-mediated colistin resistance, which may offer valuable information on the management of polymyxin resistance.

All organisms contain RNA modifications in their ribosomal RNA (rRNA), but the importance, positions and exact function of these are still not fully elucidated. Various functions such as stabilizing structures, controlling ribosome assembly and facilitating interactions have been suggested and in some cases substantiated. Bacterial rRNA contains much fewer modifications than eukaryotic rRNA. The rRNA modification patterns in bacteria differ from each other, but too few organisms have been mapped to draw general conclusions. This study maps 23S ribosomal RNA modifications in Clostridium sporogenes that can be characterized as a non-toxin producing Clostridium botulinum. Clostridia are able to sporulate and thereby survive harsh conditions, and are in general considered to be resilient to antibiotics. Selected regions of the 23S rRNA were investigated by mass spectrometry and by primer extension analysis to pinpoint modified sites and the nature of the modifications. Apparently, C. sporogenes 23S rRNA contains few modifications compared to other investigated bacteria. No modifications were identified in domain II and III of 23S rRNA. Three modifications were identified in domain IV, all of which have also been found in other organisms. Two unusual modifications were identified in domain V, methylated dihydrouridine at position U2449 and dihydrouridine at position U2500 (Escherichia coli numbering), in addition to four previously known modified positions. The enzymes responsible for the modifications were searched for in the C. sporogenes genome using BLAST with characterized enzymes as query. The search identified genes potentially coding for RNA modifying enzymes responsible for most of the found modifications.

Place: Oxford Publisher: Oxford Univ Press WOS:000574288800042; The universal L-shaped tertiary structure of tRNAs is maintained with the help of nucleotide modifications within the D- and T-loops, and these modifications are most extensive within hyperthermophilic species. The obligate-commensal Nanoarchaeum equitans and its phylogenetically-distinct host Ignicoccus hospitalis grow physically coupled under identical hyperthermic conditions. We report here two fundamentally different routes by which these archaea modify the key conserved nucleotide U54 within their tRNA T-loops. In N. equitans, this nucleotide is methylated by the S-adenosylmethionine-dependent enzyme NEQ053 to form m(5)U54, and a recombinant version of this enzyme maintains specificity for U54 in Escherichia coli. In N. equitans, m(5)U54 is subsequently thiolated to form m(5)s(2)U54. In contrast, I. hospitalis isomerizes U54 to pseudouridine prior to methylating its N1-position and thiolating the O4-position of the nucleobase to form the previously uncharacterized nucleotide m(1)s(4)Psi. The methyl and thiol groups in m(1)s(4)Psi and m(5)s(2)U are presented within the T-loop in a spatially identical manner that stabilizes the 3'-endo-anti conformation of nucleotide-54, facilitating stacking onto adjacent nucleotides and reverse-Hoogsteen pairing with nucleotide m(1)A58. Thus, two distinct structurally-equivalent solutions have evolved independently and convergently to maintain the tertiary fold of tRNAs under extreme hyperthermic conditions.

Methylation of guanosine on position N7 (m7G) on internal RNA positions has been found in all domains of life and have been implicated in human disease. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications at nucleotide resolution. In our method, m7G modified positions are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription and sequenced. We detect positions with increased mutation rates in the reduced and control samples taking the possibility of sequencing/alignment error into account and use replicates to calculate statistical significance based on log likelihood ratio tests. We show that m7G-MaP-seq efficiently detects known m7G modifications in rRNA with mutational rates up to 25% and we map a previously uncharacterised evolutionarily conserved rRNA modification at position 1581 in Arabidopsis thaliana SSU rRNA. Furthermore, we identify m7G modifications in budding yeast, human and arabidopsis tRNAs and demonstrate that m7G modification occurs before tRNA splicing. We do not find any evidence for internal m7G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA, including human Let-7e. Likewise, high sequencing depth m7G-MaP-seq analysis of mRNA from E. coli or yeast cells did not identify any internal m7G modifications.

Uracil arises in DNA by hydrolytic deamination of cytosine (C) and by erroneous incorporation of deoxyuridine monophosphate opposite adenine, where the former event is devastating by generation of C → thymine transitions. The base excision repair (BER) pathway replaces uracil by the correct base. In human cells two uracil-DNA glycosylases (UDGs) initiate BER by excising uracil from DNA; one is hSMUG1 (human single-strand-selective mono-functional UDG). We report that repair initiation by hSMUG1 involves strand incision at the uracil site resulting in a 3′-α,β-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5′-phosphate. UIP is removed from the 3′-end by human apurinic/apyrimidinic (AP) endonuclease 1 preparing for single-nucleotide insertion. hSMUG1 also incises DNA or processes UIP to a 3′-phosphate designated uracil-DNA processing product (UPP). UIP and UPP were indirectly identified and quantified by polyacrylamide gel electrophoresis and chemically characterised by matrix-assisted laser desorption/ionisation time-of-flight mass-spectrometric analysis of DNA from enzyme reactions using 18O- or 16O-water. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2′ occurs via the formation of a C1′ enolate intermediate. A three-phase kinetic model explains rapid uracil excision in phase 1, slow unspecific enzyme adsorption/desorption to DNA in phase 2 and enzyme-dependent AP site incision in phase 3.

This study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual trans-sialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid or casein glycomacropeptide (whey protein) as the sialyl donor and lactose as the acceptor, resulting in production of both 3'-sialyllactose and 6'-sialyllactose. This is the first study reporting α-2,6-trans-sialidase activity of this sialyltransferase (EC 2.4.99.1 and 2.4.99.4). A response surface design was used to evaluate the effects of three reaction parameters (pH, temperature, and lactose concentration) on enzymatic production of 3'- and 6'-sialyllactoses using 5% (w/v) casein glycomacropeptide (equivalent to 9mM bound sialic acid) as the donor. The maximum yield of 3'-sialyllactose (2.75±0.35mM) was achieved at a reaction condition with pH 6.4, 40°C, 100mM lactose after 6h; and the largest concentration of 6'-sialyllactose (3.33±0.38mM) was achieved under a condition with pH 5.4, 40°C, 100mM lactose after 8h. 6'-sialyllactose was presumably formed from α-2,3 bound sialic acid in the casein glycomacropeptide as well as from 3'-sialyllactose produced in the reaction. The kcat/Km value for the enzyme using 3'-sialyllactose as the donor for 6'-sialyllactose synthesis at pH 5.4 and 40°C was determined to be 23.22±0.7M(-1)s(-1). Moreover, the enzyme was capable of catalyzing the synthesis of both 3'- and 6'-sialylated galactooligosaccharides, when galactooligosaccharides served as acceptors.

Guanosines are important for biological activities through their specific functional groups that are recognized for RNA or protein interactions. One example is recognition of N1 of G37 in tRNA by S-adenosyl-methionine (AdoMet)–dependent tRNA methyltransferases to synthesize m1G37-tRNA, which is essential for translational fidelity in all biological domains. Synthesis of m1G37-tRNA is catalyzed by TrmD in bacteria and by Trm5 in eukarya and archaea, using unrelated and dissimilar structural folds. This raises the question of how dissimilar proteins recognize the same guanosine. Here we probe the mechanism of discrimination among functional groups of guanosine by TrmD and Trm5. Guanosine analogs were systematically introduced into tRNA through a combination of chemical and enzymatic synthesis. Single turnover kinetic assays and thermodynamic analysis of the effect of each analog on m1G37-tRNA synthesis reveal that TrmD and Trm5 discriminate functional groups differently. While both recognize N1 and O6 of G37, TrmD places a much stronger emphasis on these functional groups than Trm5. While the exocyclic 2-amino group of G37 is important for TrmD, it is dispensable for Trm5. In addition, while an adjacent G36 is obligatory for TrmD, it is nonessential for Trm5. These results depict a more rigid requirement of guanosine functional groups for TrmD than for Trm5. However, the sensitivity of both enzymes to analog substitutions, together with an experimental revelation of their low cellular concentrations relative to tRNA substrates, suggests a model in which these enzymes rapidly screen tRNA by direct recognition of G37 in order to monitor the global state of m1G37-tRNA.

tRNA is the most heavily modified of all RNA types, with typically 10–20% of the residues being post-transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications [1], many of which are chemically similar. Furthermore, the tRNA has to be investigated with single-nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNALys(UUU) from Trypanosoma brucei, and provide a complete overview of its post-transcriptional modifications. The first step was MALDI-TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass-changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence of the unmodified tRNA revealed the modified residues. The modifications were further characterized at the nucleoside level by chromatographic retention time and fragmentation pattern upon higher-order tandem MS. Phylogenetic comparison with modifications in tRNALys from other organisms was used through the entire analysis. We identified modifications on 12 nucleosides in tRNALys(UUU), where U47 exhibited a novel modification, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, based on identical chromatographic retention and MS fragmentation as the synthetic nucleoside. A37 was observed in two versions: a minor fraction with the previously described 2-methylthio-N6-threonylcarbamoyl-modification, and a major fraction with A37 being modified by a 294.0-Da moiety. The latter product is the largest adenosine modification reported so far, and we discuss its nature and origin.

Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm5U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8−/− mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm5U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2′-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) and 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8−/− mice appear normal. However, the selenocysteine-specific tRNA (tRNASec) is aberrantly modified in the Alkbh8−/− mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.

The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfr− strains with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m2A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position and is independent of methylation at the 2 position of A2503. This investigation is, to our knowledge, the first instance where the 8-methyladenosine modification has been described in natural RNA molecules.

A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant trans-sialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heterogeneous high-mannose type N-glycans were present at all the five potential N-linked glycosites. N-linked glycans in Tr7 were predominantly neutral oligosaccharides with compositions Man(8-16)GlcNA(c2), but also mono- and di-phosphorylated oligosaccharides in the forms of Man(9-15)P(1)GlcNA(c2) and Man(9-14)P(2)GlcNA(c2), respectively. Some phosphorylated N-linked glycans further contained an additional HexNAc, which has not previously been reported in P. pastoris-expressed proteins. We compiled a method pipeline that combined hydrophilic interaction liquid chromatography enrichment of glycopeptides, high accuracy mass spectrometry and automated interpretation of the mass spectra with in-house developed "MassAI" software, which proved efficient in glycan site microheterogeneity analysis. Functional analysis showed that the deglycosylated Tr7 retained more than 90% of both the sialidase and trans-sialidase activities relative to the glycosylated Tr7.

Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3'- and 6'-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5'-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6'-sialyllactose synthesis but low levels of 3'-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmSTWT under optimal conditions for 6'-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3'-sialyllactose over 6'-sialyllactose and the double mutant (PmSTE271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmSTE271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.

To reveal the gas-phase chemistry of RNA and DNA fragmentation during MALDI mass spectrometry in positive ion mode, we performed hydrogen/deuterium exchange on a series of RNA and DNA tetranucleotides and studied their fragmentation patterns on a high-resolution MALDI TOF-TOF instrument. We were specifically interested in elucidating the remarkably different fragmentation behavior of RNA and DNA, i.e., the characteristic and abundant production of c- and y-ions from RNA versus a dominating generation of (a-B)- and w-ions from DNA analytes. The analysis yielded important information on all significant backbone cleavages as well as nucleobase losses. Based on this, we suggest common fragmentation mechanisms for RNA and DNA as well as an important RNA-specific reaction requiring a 2′-hydroxyl group, leading to c- and y-ions. The data is viewed and discussed in the context of previously published data to obtain a coherent picture of the fragmentation of singly protonated nucleic acids.

A novel single-base extension (SBE) assay using cleavable and noncleavable SBE primers in the same reaction mix is described. The cleavable SBE primers consisted of deoxyribonucleotides and one ribonucleotide (hereafter denoted chimeric primers), whereas the noncleavable SBE primers consisted of only deoxyribonucleotides (hereafter denoted standard primers). Biotin-labeled ddNTPs were used in the SBE reaction, and the SBE products were purified using the monomeric avidin triethylamine purification protocol, ensuring that only primers extended with a biotin-ddNTP in the 3'-end were isolated. A ribonuclease mix was developed to specifically cleave the chimeric primers, irrespective of the base of the ribonucleotide, whereas standard primers without a ribonucleotide were unaffected by the ribonuclease treatment. The SBE products were analyzed in linear mode using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The cleaved SBE products were detected in the 2000-5500 m/z range, and the noncleaved SBE products were detected in the 5500-10 000 m/z range. The method was validated by typing 17 Y chromosome single-nucleotide polymorphisms in 100 males with a 17-plex SBE package containing 9 chimeric primers and 8 standard primers.

One of the most promising techniques for typing of multiple single-nucleotide polymorphism (SNP) is detection of single base extension primers (SBE) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a new MALDI-TOF MS protocol for typing of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle conditions using 5% triethylamine. We dubbed this method monomeric avidin triethylamine purification. The biotin-labeled ddNTPs contained linkers with different masses ensuring a clear separation of the alleles even for SBE primers with a mass of 10 300 Da. Furthermore, only 25-350 fmol of SBE primers were necessary in order to obtain reproducible MALDI-TOF spectra. Similar signal intensities were obtained in the 5500-10 300 m/z mass range by increasing the concentration of the longer SBE primers in the reaction. To validate the technique, 17 Y-chromosome SNPs were analyzed in 200 males. The precision and accuracy of the mass determination were analyzed by parametric statistic, and the potential use of MALDI-TOF MS for SNP typing is discussed.

Rigid linear and tripoidal organic modules based on the oligo(phenylene ethynylene) backbone having salicylaldehyde-derived termini are synthesized. A highly functionalized 5-iodosalicyl aldehyde was prepared and coupled to each ethynyl group of 1,4-diethynylbenzene or 1,3,5-triethynylbenzene in Sonogashira couplings. The two or three termini of the compounds are functionalized for incorporation in linear and branched oligonucleotide strands. For the linear module (LM), the two termini are equipped with amide spacers, and one of these was functionalized with a DMTr (dimethoxytrityl)-protected hydroxy group and the other with a phosphoramidite. One of the tripoidal modules is prepared with DMTr groups in two of its three termini. A tripoidal module is also synthesized with three different groups on its hydroxy termini: a phosphoramidite, a DMTr group, and an Fmoc group. Extended studies have shown that these rigid linear and tripoidal organic modules can be incorporated into short oligonucleotides. Several of these modules can be applied for DNA-directed assembly and covalent coupling into structures of predetermined connectivity. Such structures have potential application for molecular electronics and nanotechnology.

There are three sites of m(5)U modification in Escherichia coli stable RNAs: one at the invariant tRNA position U54 and two in 23S rRNA at the phylogenetically conserved positions U747 and U1939. Each of these sites is modified by its own methyltransferase, and the tRNA methyltransferase, TrmA, is well-characterised. Two open reading frames, YbjF and YgcA, are approximately 30% identical to TrmA, and here we determine the functions of these candidate methyltransferases using MALDI mass spectrometry. A purified recombinant version of YgcA retains its activity and specificity, and methylates U1939 in an RNA transcript in vitro. We were unable to generate a recombinant version of YbjF that retained in vitro activity, so the function of this enzyme was defined in vivo by engineering a ybjF knockout strain. Comparison of the methylation patterns in 23S rRNAs from YbjF(+) and YbjF(-) strains showed that the latter differed only in the lack of the m(5)U747 modification. With this report, the functions of all the E.coli m(5)U RNA methyltransferases are identified, and a more appropriate designation for YbjF would be RumB (RNA uridine methyltransferases B), in line with the recent nomenclature change for YgcA (now RumA).

Avilamycin is an orthosomycin antibiotic that has shown considerable potential for clinical use, although it is presently used as a growth promoter in animal feed. Avilamycin inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit. The ribosomes of the producer strain, Streptomyces viridochromogenes Tu57, are protected from the drug by the action of three resistance factors located in the avilamycin biosynthetic gene cluster. Two of the resistance factors, aviRa and aviRb, encode rRNA methyltransferases that specifically target 23S rRNA. Recombinant AviRa and AviRb proteins retain their activity after purification, and both specifically methylate in vitro transcripts of 23S rRNA domain V. Reverse transcriptase primer extension indicated that AviRa is an N-methyltransferase that targets G2535 within helix 91 of the rRNA, whereas AviRb modified the 2'-O-ribose position of nucleotide U2479 within helix 89. MALDI mass spectrometry confirmed the exact positions of each of these modifications, and additionally established that a single methyl group is added at each nucleotide. Neither of these two nucleotides have previously been described as a target for enzymatic methylation. Molecular models of the 50S subunit crystal structure show that the N-1 of the G2535 base and the 2'-hydroxyl of U2479 are separated by approximately 10 A, a distance that can be spanned by avilamycin. In addition to defining new resistance mechanisms, these data refine our understanding of the probable ribosome contacts made by orthosomycins and of how these antibiotics inhibit protein synthesis.

We have studied the fragmentation behaviour of short, singly protonated oligoribonucleotides on a MALDI Qq-TOF instrument with the aim of using this instrumental set-up to characterise modifications of RNA molecules. Individual ion species from enzymatically generated mixtures were isolated in one quadrupole and subjected to collision-induced dissociation in a second quadrupole followed by separation of the resulting product ions in an orthogonal time-of-flight mass analyser. Complex spectra were generally observed with nearly all types of cleavages along the phosphodiester backbone and of the N-glycosidic bonds (and combinations of these) occurring, albeit at different relative intensities. The most labile part of the backbone was found to be the 5′-P-O bond, resulting in c- and y-ions. Loss of neutral cytosine and guanine occurred equally often, whereas neutral loss of adenosine was less prevalent. Loss of uracil, either neutral or charged species, was not observed. Because the fragmentation pattern observed here is significantly different from what has been reported for singly protonated oligodeoxyribonucleotides, we suggest that the 2′-substituent in the sugar plays a central role in the fragmentation mechanisms of nucleic acids. Finally, we used the acquired knowledge about oligoribonucleotide fragmentation to characterise an in vivo methylated oligoribonucleotide by tandem mass spectrometry. Copyright © 2001 John Wiley & Sons, Ltd.

Double-stranded DNA ranging from 9 kDa to over 500 kDa were desorbed and analyzed by MALDI TOF mass spectrometry. IR-MALDI with glycerol as matrix yielded excellent results for larger double-stranded DNA by adjustment of the ionic strength through the addition of salts. Very little fragmentation and a routine sensitivity in the subpicomole range were observed in IR-MALDI when double-stranded analytes harboring 70 base pairs or more were probed. In the lower mass range (up to approximately 70 base pairs), UV-MALDI with 6-aza-2-thiothymine as matrix was the ionization method of choice because it allowed specific double-stranded complexes containing relatively few base pairs to be desorbed intactly. In this mode, an essentially quantitative detection of the double-stranded form was observed for a 70-mer. The UV-MALDI was accompanied by a significant fragmentation and a resulting reduced sensitivity and mass resolution. The methods described open MALDI-MS for the analysis of large DNA-DNA and DNA-protein complexes.

Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10 7 U M −1. The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn 2+ addition caused regain of the activity, but Zn 2+addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1′ at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

Casein glycomacropeptide (cGMP) and lactose, which are purified (or semi-purified) components obtained from side streams from dairy industry operations, were used as substrates for enzyme catalyzed production of 3′-sialyllactose, a model case compound for human milk oligosaccharides (HMOs). The enzyme employed was a mutated sialidase, Tr6, derived from Trypanosoma rangeli , and expressed in Pichia pastoris after codon-optimization. The Tr6 contained 6 point mutations and exhibited trans -sialidase activity. The Tr6 trans -sialidase reaction conditions were tuned for maximizing Tr6 catalyzed 3′-sialyllactose production by optimizing pH, temperature, acceptor, and donor concentrations using response surface designs. At the optimum reaction conditions, the Tr6 catalyzed the transfer of sialic acid from cGMP to lactose at high efficiency without substantial hydrolysis of the 3′-sialyllactose product. The robustness of the Tr6 catalyzed reaction was verified at 5 L-scale providing a yield of 3.6 g 3′-sialyllactose at an estimated molar trans -sialylation yield of 50% on the 3′-sialyl in cGMP. Lacto- N -tetraose and lacto- N -fucopentaoses also functioned as acceptor molecules demonstrating the versatility of the Tr6 trans -sialidase for catalyzing sialyl-transfer for generating different HMOs. The data signify the applicability of enzymatic trans -sialylation on dairy side-stream components for production of human milk oligosaccharides.

The determination of RNA sequences using base- specific enzymatic cleavages is a well established method. Different synthetic RNA molecules were analyzed for uniformity of degradation by RNase T1, U2, A and PhyM under reaction conditions compatible with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), to identify the positions of G, A and pyrimidine residues. In order to get a complete set of fragments derived from cleavage at every phosphodiester bond, the samples were also subjected to a limited alkaline hydrolysis. Additionally, the 5'-terminus fragments of a 49mer RNA transcript were isolated by way of 5'-biotinylation and streptavidin-coated magnetic beads (Dynal), followed by a RNase U2digestion. MALDI-MS of the generated fragments is presented as an efficient technique for a direct read out of the nucleotide sequence.

The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the RlmN methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance. The enzymes are homologous and use the same mechanism involving radical S -adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine in the enzyme and a transient cross-linking to the RNA, but they differ in which carbon atom in the adenine they methylate. Comparative sequence analysis identifies differentially conserved residues that indicate functional sequence divergence between the two classes of Cfr- and RlmN-like sequences. The differentiation between the two classes is supported by previous and new experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. Finally, evolutionary aspects of the distribution of Cfr- and RlmN-like enzymes are discussed.

Here we show that the ubiquitin-conjugating enzyme Rad6p plays a crucial role in locus-specific replacement recombination in the TRP1-ARS1 region. In rad6-1 strains, where this ubiquitination activity is modified, homologous recombination across a 150 bp continuous region is completely abolished. Our results unambiguously identified the ARS1 scaffold attached region (SAR) as being the region where this impediment for replacement recombination is located, since a merging of the location of the recombination impediment and binding properties in a scaffold exchange assay with deletion mutations was observed. Our observations strongly support the notion of torsionally separated chromosomal domains being organized by SARs and scaffold proteins, and being dynamically realigned as a consequence of ubiquitination and proteolysis.

The present article is a survey of ESI and MALDI mass spectrometric analysis of nucleic acid oligomers and polymers. In order to limit the extent of the review, mass spectrometry of mononucleotides is generally not considered, except where such data are important for an understanding of the analysis of larger nucleic acids. The first part of the review is a condensed description of the structure and the acid-base properties of nucleic acids. The remaining part is divided into three main sections, dealing with the practical aspects of the two ionization techniques, fragmentation, and applications, respectively. The first section includes an extensive discussion of experimental parameters and problems, which are important for the analysis of different types of nucleic acid samples, including noncovalent complexes and mixtures. At the end of this section, as well as the following one, a comparison between MALDI and ESI as ionization techniques for nucleic acid is given. In addition to a detailed discussion of ion fragmentation, the fragmentation section includes an overview of the direct mass spectrometric sequencing of nucleic acids performed with either technique. The fragmentation reactions occurring upon MALDI and ESI are compared. The last section describes the life science applications of ESI-MS and MALDI-MS of nucleic acids; an account of experiments demonstrating the potential of a method, and of the bona fide solving of problems by ESI and MALDI is given. © 1997 John Wiley & Sons, Inc.

Automated synthesis of novel mono-branched (“Y-shaped”) and double-branched (“H-shaped”) oligodeoxynucleotide analogues has been accomplished by use of phosphoramidite chemistry. The synthetic strategy involves stereoselective 3′- O -phosphitylation of nucleoside 2 to give building block 3 and fully automated stereoselective elongation and subsequent branching of amidite 3 on a DNA-synthesizer. Thus, this strategy allows construction of branched nucleic acid molecules with sequences of arbitrary branch length and base composition. Compared to the corresponding linear analogues, the novel branched oligodeoxynucleotides exhibited increased thermal stability in intramolecular and intermolecular hybridization experiments.

UV and IR matrix-assisted laser desorption/ionization mass spectrometry (UV- and IR-MALDI) have demonstrated their potential for the accurate and sensitive mass determination of oligonucleotides. Metastable molecule ion fragmentation was found to be the main limitation in both desorption schemes for the analysis of larger nucleic acid sequences. Fragment ions from additional prompt decays, observed only in IR-MALDI, offer structural data, which allow sequence information to be derived for low-picomole amounts of oligodeoxynucleotides with up to 21 bases from a single mass spectrum. Two examples demonstrating the feasibility of this new sequencing technique are given. A model is introduced and discussed, which proposes a reaction scheme for the observed fragment ion patterns of nucleic acids and differentiates prompt and metastable fragmentation mechanisms. The role of fragmentation in direct mass spectrometric sequencing schemes and as a limitation for the accessible mass range in nucleic acid MALDI mass spectrometry is discussed.

The palindromic deoxyribonucleotides 5'-CGCGA-TATCGCG-3' and 5'-CGCGTTAACGCG-3' have been characterized by 1H NMR spectroscopy. The NMR data identified both B-DNA duplex conformations and hairpin conformations, the latter with loop regions consisting of the four central nucleotides. The resonances of the various conformations were assigned by use of two-dimensional NMR methods. The relative stability of the various conformations was investigated as a function of temperature, ionic strength and nucleotide concentration. The duplexes were found to be stabilized at high ionic strength and at low temperature, while the hairpins were stabilized at low ionic strength and at medium temperature. The thermodynamics of the duplex-hairpin and the hairpin-random coil transitions were examined, and compared to the other two oligonucleotide in the palindromic d(CGCG(A/T)4CGCG) oligonucleotide family. The relative stabilities of the duplex conformations with respect to the random coil conformations are similar for the d(CGCGAATTCGCG), d(CGCGATATCGCG) and d(CGCGTATACGCG) oligonucleotides. The duplex conformation of d(CGCGTTAACGCG) is less stable. The hairpin of d(CGCGTTAACGCG) seems also to be less stable relative to the random coil conformation than in the case of the other oligonucleotides at an equal oligonucleotide concentration. A cruciform intermediate between the duplex and hairpin conformations is suggested to explain some discrepancies observed in this work in case of the d(CGCGTTAACGCG) oligonucleotide. This is similar to what has been reported for the d(CGCGTATACGCG) oligonucleotide.

Many powerful analytical techniques for investigation of nucleic acids exist in the average modern molecular biology lab. The current review will focus on questions in RNA biology that have been answered by the use of mass spectrometry, which means that new biological information is the purpose and outcome of most of the studies we refer to. The review begins with a brief account of the subject "MS in the biology of RNA" and an overview of the prevalent RNA modifications identified to date. Fundamental considerations about mass spectrometric analysis of RNA are presented with the aim of detailing the analytical possibilities and challenges relating to the unique chemical nature of nucleic acids. The main biological topics covered are RNA modifications and the enzymes that perform the modifications. Modifications of RNA are essential in biology, and it is a field where mass spectrometry clearly adds knowledge of biological importance compared to traditional methods used in nucleic acid research. The biological applications are divided into analyses exclusively performed at the building block (mainly nucleoside) level and investigations involving mass spectrometry at the oligonucleotide level. We conclude the review discussing aspects of RNA identification and quantifications, which are upcoming fields for MS in RNA research. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

Novel flexible ollgodeoxynucleotide analogues containing (R)- and (S)-3′,4′-seco-thymidine were synthesized on an automated DNA-synthesizer using the phosphoramidlte approach. Oligodeoxynucleotide analogues (17-mers) having one or three modifications In the middle or one or two modifications In the ends were evaluated with respect to hybridization properties and enzymatic stability. 3′-End-modlfled oligomers were stable towards 3′-exonuclease degradation and displayed acceptable hybridization properties.

tRNA is the most heavily modified of all RNA types, with typically 10-20% of the residues being post-transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications, many of which are chemically similar. Furthermore, the tRNA has to be investigated with single-nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNA(Lys)(UUU) from Trypanosoma brucei, and provide a complete overview of its post-transcriptional modifications. The first step was MALDI-TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass-changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence of the unmodified tRNA revealed the modified residues. The modifications were further characterized at the nucleoside level by chromatographic retention time and fragmentation pattern upon higher-order tandem MS. Phylogenetic comparison with modifications in tRNA(Lys) from other organisms was used through the entire analysis. We identified modifications on 12 nucleosides in tRNA(Lys)(UUU), where U47 exhibited a novel modification, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, based on identical chromatographic retention and MS fragmentation as the synthetic nucleoside. A37 was observed in two versions: a minor fraction with the previously described 2-methylthio-N(6)-threonylcarbamoyl-modification, and a major fraction with A37 being modified by a 294.0-Da moiety. The latter product is the largest adenosine modification reported so far, and we discuss its nature and origin.

Knowledge concerning the molecular mechanisms governing the influence of non-coding RNAs on protein production has emerged rapidly during the past decade. Today, two main research areas can be identified, one oriented toward the use of artificially introduced siRNAs for manipulation of gene expression, and the other one focused on the function of endogenous miRNAs. In both cases, the active molecule consists of a ∼20-nucleotide-long RNA duplex. In the siRNA case, improved systemic stability is of central interest for its further development toward clinical applications. With respect to miRNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized by a combination of gel electrophoresis and MALDI-MS techniques, and the silencing capacity was evaluated in cellular luciferase-expressing systems using HB2 cells. Data show that platination of the antisense strand of the siRNAs results in adducts with protection against hydrolytic cleavage in the proximity of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays all confirm biological activity of antisense-platinated siRNAs, here with platination sites located outside of the seed region. A significant reduction of silencing capacity was observed as a general trend, however. Of the two complexes studied, oxaliplatin exhibits the larger influence, thus indicating subtle differences between the abilities of cis- and oxaliplatin to interfere with si- and miRNA processing.

Erm methyltransferases are prevalent in pathogenic bacteria and confer resistance to macrolide, lincosamide, and streptogramin B antibiotics by specifically methylating the 23S ribosomal RNA at nucleotide A2058. We have identified motifs within the rRNA substrate that are required for methylation by Erm. Substrate molecules were constructed in a combinatorial manner from two separate sets (top and bottom strands) of short RNA sequences. Modifications, including LNA monomers with locked sugar residues, were incorporated into the substrates to stabilize their structures. In functional substrates, the A2058 methylation target (on the 13- to 19-nucleotide top strand) was displayed in an unpaired sequence immediately following a conserved irregular helix, and these are the specific structural features recognized by Erm. Erm methylation was enhanced by stabilizing the top-strand conformation with an LNA residue at G2056. The bottom strand (nine to 19 nucleotides in length) was required for methylation and was still functional after extensive modification, including substitution with a DNA sequence. Although it remains possible that Erm makes some unspecific contact with the bottom strand, the main role played by the bottom strand appears to be in maintaining the conformation of the top strand. The addition of multiple LNA residues to the top strand impeded methylation; this indicates that the RNA substrate requires a certain amount of flexibility for accommodation into the active site of Erm. The combinatorial approach for identifying small but functional RNA substrates is a step towards making RNA-Erm complexes suitable for cocrystal determination, and for designing molecules that might block the substrate-recognition site of the enzyme.

Complete characterization of a biomolecule's chemical structure is crucial in the full understanding of the relations between their structure and function. The dominating components in ribosomes are ribosomal RNAs (rRNAs), and the entire rRNA—but a single modified nucleoside at position 2501 in 23S rRNA—has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown modification as 5-hydroxycytidine—a novel modification in RNA. Identification of 5-hydroxycytidine was completed by liquid chromatography under nonoxidizing conditions using a graphitized carbon stationary phase in combination with ion trap tandem mass spectrometry and by comparing the fragmentation behavior of the natural nucleoside with that of a chemically synthesized ditto. Furthermore, we show that 5-hydroxycytidine is also present in the equivalent position of 23S rRNA from the bacterium Deinococcus radiodurans. Given the unstable nature of 5-hydroxycytidine, this modification might be found in other RNAs when applying the proper analytical conditions as described here.

Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA ( tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and (S)-5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA(UCG)(Arg), and tRNA(UCC)(Gly), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into ( S)- mchm(5)U in tRNAUCCGly. These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.

Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm 5 U), 5-carbamoylmethyluridine (ncm 5 U) or derivatives thereof. Here, we demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm 5 U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm 5 U to ( S ) - mchm 5 U in tRNA GlyUCC , and has a function similar to the mammalian dioxygenase ALKBH8. Interestingly, atalkbh8 mutant plants displayed strongly increased levels of mcm 5 U, and also of mcm 5 Um, its 2′- O -ribose methylated derivative. This suggests that accumulated mcm 5 U is prone to further ribose methylation by a non-specialized mechanism, and may challenge the notion that the existence of mcm 5 U- and mcm 5 Um-containing forms of the selenocysteine-specific tRNA Sec in mammals reflects an important regulatory process. The present study reveals a role in for several hitherto uncharacterized Arabidopsis proteins in the formation of modified wobble uridines. © The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with infrared laser light of a wavelength of 2.94 microns has been used for the analysis of nucleic acids. Spectra of oligodeoxynucleotides up to 26 nucleotides, oligothymidylic acids up to 100 nucleotides as well as different synthetic RNA oligomers and RNA transcripts up to 104 nucleotides are presented. A main problem in the analysis of oligodeoxynucleotides was found to be related to the loss of bases. The stability of oligothymidylic acids as opposed to oligodeoxynucleotides containing all four bases indicates that the loss of bases is correlated with A, C and G protonation which decreases the stability of the N-glycosidic bond. Experiments indicate that the breakage of the N-glycosidic bond probably occurs during the desorption process due to proton transfer from the phosphodiester groups to the ionizable bases. RNA displayed a significantly higher stability in MALDI-MS due to the presence of a 2'-OH group. Consequently, signals of RNA transcripts with a length of up to 142 nucleotides could be detected by MALDI-MS. Technical details of the method, including the distribution of positive counterions on the phosphodiester backbone, the upper mass limit and mass accuracy are discussed along with a number of potential analytical applications.

Udgivelsesdato: 2009-Apr The idea of identifying or characterizing an RNA molecule based on a mass spectrum of specifically generated RNA fragments has been used in various forms for well over a decade. We have developed software-named RRM for 'RNA mass mapping'-which can search whole prokaryotic genomes or RNA FASTA sequence databases to identify the origin of a given RNA based on a mass spectrum of RNA fragments. As input, the program uses the masses of specific RNase cleavage of the RNA under investigation. RNase T1 digestion is used here as a demonstration of the usability of the method for RNA identification. The concept for identification is that the masses of the digestion products constitute a specific fingerprint, which characterize the given RNA. The search algorithm is based on the same principles as those used in peptide mass fingerprinting, but has here been extended to work for both RNA sequence databases and for genome searches. A simple and powerful probability model for ranking RNA matches is proposed. We demonstrate viability of the entire setup by identifying the DNA template of a series of RNAs of biological and of in vitro transcriptional origin in complete microbial genomes and by identifying authentic 16S ribosomal RNAs in a 'small ribosomal subunit RNA' database. Thus, we present a new tool for a rapid identification of unknown RNAs using only a few picomoles of starting material.

Resistance to antibiotics that target the bacterial ribosome is often conferred by methylation at specific nucleotides in the rRNA. The nucleotides that become methylated are invariably key sites of antibiotic interaction. The addition of methyl groups to each of these nucleotides is catalyzed by a specific methyltransferase enzyme. The Erm methyltransferases are a clinically prevalent group of enzymes that confer resistance to the therapeutically important macrolide, lincosamide, and streptogramin B (MLS B) antibiotics. The target for Erm methyltransferases is at nucleotide A2058 in 23S rRNA, and methylation occurs before the rRNA has been assembled into 50S ribosomal particles. Erm methyltransferases occur in a phylogenetically wide range of bacteria and differ in whether they add one or two methyl groups to the A2058 target. The dimethylated rRNA confers a more extensive MLS B resistance phenotype. We describe here a method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to determine the location and number of methyl groups added at any site in the rRNA. The method is particularly suited to studying in vitro methylation of RNA transcripts by resistance methyltransferases such as Erm.

Resistance to antibiotics that target the bacterial ribosome is often conferred by methylation at specific nucleotides in the rRNA. The nucleotides that become methylated are invariably key sites of antibiotic interaction. The addition of methyl groups to each of these nucleotides is catalyzed by a specific methyltransferase enzyme. The Erm methyltransferases are a clinically prevalent group of enzymes that confer resistance to the therapeutically important macrolide, lincosamide, and streptogramin B (MLS B) antibiotics. The target for Erm methyltransferases is at nucleotide A2058 in 23S rRNA, and methylation occurs before the rRNA has been assembled into 50S ribosomal particles. Erm methyltransferases occur in a phylogenetically wide range of bacteria and differ in whether they add one or two methyl groups to the A2058 target. The dimethylated rRNA confers a more extensive MLS B resistance phenotype. We describe here a method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to determine the location and number of methyl groups added at any site in the rRNA. The method is particularly suited to studying in vitro methylation of RNA transcripts by resistance methyltransferases such as Erm.

Posttranscriptional modifications on the base or sugar of ribonucleosides generally result in mass increases that can be measured by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a direct and accurate means of determining the masses of RNAs. Mass spectra produced by MALDI are relatively straightforward to interpret, because they are dominated by singly charged ions, making it possible to analyze complex mixtures of RNA oligonucleotides ranging from trinucleotides up to 20-mers. Analysis of modifications within much longer RNAs, such as ribosomal RNAs, can be achieved by digesting the RNA with nucleotide-specific enzymes. In some cases, it may be desirable to isolate specific sequence regions before MALDI-MS analysis, and this requires a few additional steps. The method is applicable to the study of modified RNAs from cell extracts as well as RNA modifications added in cell-free in vitro systems. MALDI-MS is particularly useful in cases in which other techniques such as those involving primer extension or chromatographic analyses are not practicable. To date, MALDI-MS has been used to localize rRNA modifications that are involved in fundamental processes in protein synthesis as well as methylations that confer resistance to antibiotics. For several rRNA sites, MALDI-MS has served an essential role in the identification of the enzymes that catalyze the modifications.

Posttranscriptional modifications on the base or sugar of ribonucleosides generally result in mass increases that can be measured by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a direct and accurate means of determining the masses of RNAs. Mass spectra produced by MALDI are relatively straightforward to interpret, because they are dominated by singly charged ions, making it possible to analyze complex mixtures of RNA oligonucleotides ranging from trinucleotides up to 20-mers. Analysis of modifications within much longer RNAs, such as ribosomal RNAs, can be achieved by digesting the RNA with nucleotide-specific enzymes. In some cases, it may be desirable to isolate specific sequence regions before MALDI-MS analysis, and this requires a few additional steps. The method is applicable to the study of modified RNAs from cell extracts as well as RNA modifications added in cell-free in vitro systems. MALDI-MS is particularly useful in cases in which other techniques such as those involving primer extension or chromatographic analyses are not practicable. To date, MALDI-MS has been used to localize rRNA modifications that are involved in fundamental processes in protein synthesis as well as methylations that confer resistance to antibiotics. For several rRNA sites, MALDI-MS has served an essential role in the identification of the enzymes that catalyze the modifications.

Mass spectrometry has become an increasingly important tool of high accuracy, efficiency, and speed for the routine analysis of nucleic acids. To make it useful for large-scale sequencing of genomic material as required for example in genotyping and clinical diagnosis, it is necessary to find approaches that allow the analysis of sequences much larger than the 100 nucleotides currently possible. Matrix-assisted laser desorption/ionization (MALDI) mass spectra of synthetic DNA, restriction enzyme fragments of plasmid DNA, and RNA transcripts up to a size of 2180 nucleotides are reported. The demonstrated mass accuracy of 1 percent or better and the sample requirement of a few femtomoles or less surpass all currently available techniques for the analysis of large nucleic acids. DNA and RNA can be analyzed with only a limited investment in sample purification.

The sequence and the structure of DNA methyltransferase-2 (Dnmt2) bear close affinities to authentic DNA cytosine methyltransferases. A combined genetic and biochemical approach revealed that human DNMT2 did not methylate DNA but instead methylated a small RNA; mass spectrometry showed that this RNA is aspartic acid transfer RNA (tRNA Asp ) and that DNMT2 specifically methylated cytosine 38 in the anticodon loop. The function of DNMT2 is highly conserved, and human DNMT2 protein restored methylation in vitro to tRNA Asp from Dnmt2-deficient strains of mouse, Arabidopsis thaliana, and Drosophila melanogaster in a manner that was dependent on preexisting patterns of modified nucleosides. Indirect sequence recognition is also a feature of eukaryotic DNA methyltransferases, which may have arisen from a Dnmt2-like RNA methyltransferase.