Estimation of in vivo aminoacylation by periodate oxidation: tRNA alterations and iodate inhibition.

Two problems associated with periodate oxidation in determining the extent of aminoacylation of tRNA are discussed. One of the products of this reaction, sodium iodate, was found to inhibit tRNA charging. In addition, periodate oxidation also appears to alter sites other than the 3′-end on at least two isoacceptor species of tRNA^Leu.     

Model system oxidations supporting the crystal growth model of ferritin iron uptake

Fe²⁺ is oxidized and taken up by ferritin or ápoferritin in the presence of dioxygen. Iodate causes Fe²⁺ oxidation and uptake by ferritin, but not by apoferritin. Synthetic iron polymer facilitates Fe²⁺ oxidation by either dioxygen or iodate. Nitrilotriacetic acid or iminodiacetic acid facilitate oxidation of Fe²⁺ by oxygen but not by iodate. These results support the crystal growth model of ferritin iron uptake, with iron polymer serving as a model for the ferritin core and aminocarboxylic acids mimicking the metal-binding sites of apoferritin.     

Presence and Function of Sulfur-containing Transfer Ribonucleic Acid of Bacillus subtilis

Bacillus subtilis transfer ribonucleic acid (tRNA) was analyzed for the occurrence of thionucleotides by in vivo labeling with (35)S and fractionation by methylated albumin kieselguhr column chromatography. Alkaline hydrolysates of tRNA were also examined by column chromatography and paper electrophoresis, and the amino acid-accepting ability of thionucleotide-containing tRNA was tested after iodine oxidation. The results showed that B. subtilis tRNA contains 4-thiouridylate, a second nucleotide with properties similar to 2-thiopyrimidine, and a third unidentified thionucleotide. The amino acid-accepting ability for serine, tyrosine, lysine, and glutamic acid was markedly inhibited after oxidation of the tRNA with iodine, suggesting the presence of thionucleotides in these tRNA species. This inhibition could be reversed by thiosulfate reduction. The iodine treatment totally inactivated all lysine tRNA species, partially inactivated the serine tRNA species, and did not affect the accepting ability for valine. A comparison of tRNA from cells in the log and stationary phases and from spores revealed similar iodine inactivation patterns in all cases. The thionucleotide content in B. subtilis tRNA differed from that in Escherichia coli, both in extent and in distribution. A possible function of the thionucleotides in tRNA is discussed.     

Transfer RNATyr of melanoma tissues and cells: relevance to melanin synthesis?

The tRNA present in swine melanoma tumor tissue and normal gray skin tissue were compared by aminoacylation of the unfractionated tRNA preparations. Of the seventeen amino acids studied, seven showed differences in rate of acceptance to tRNAs from normal and tumor tissues; the tRNAs of two amino acids, tyrosine and glycine, showed dramatic three fold increases in melanoma tumor. As melanin biosynthesis proceeds from tyrosine oxidation the investigations focused on the increase in tyrosine tRNA. Kinetic analysis of tyrosine aminoacylation to normal and melanoma tRNAs revealed no differences. Analysis of the isoaccepting species of tRNATyr from normal skin and melanoma tumor tissues identified three isoacceptors; tRNATyr, represented the predominant species in normal gray skin, while tRNA2Tyr predominated in melanoma tumor tissue. The tyrosine acceptances by tRNAs from three human melanoma cell lines were analyzed and found to be variable, but isoaccepting species analysis of the tRNATyr of these three cell lines still showed a correlation between the preponderance of tRNA2Tyr and extent of tyrosine acceptance. Additionally the enzymatic activity for the oxidation of tyrosine was found to be related to tyrosine acceptance and tRNA2Tyr predominance..     

Selective 32P-labelling of individual species in a total tRNA population.

A simple procedure to label individual tRNA species in a total tRNA preparation has been developed. The principle of the method is as follows: total crude tRNA (from E. coli) is incubated in the presence of a crude aminoacyl-tRNA synthetase preparation, containing most aminoacyl-tRNA synthetases and only one specific amino acid corresponding to the tRNA species which is intended to be labelled. This achieves the purpose of charging the desired tRNA species thereby protecting its 3'OH-terminus; obviously all the other tRNA species will have a free 3'OH group. Periodate oxidation, followed by beta-elimination, destroys any free 3'OH. After deacylation of the specific aminoacylated tRNA at pH 8.8 the only free 3'OH group will be the one of the desired tRNA species. High specific activity (32P)-pCp is ligated to this 3'OH by means of T4-RNA ligase. Two-dimensional polyacrylamide gel electrophoresis (2D-PGE) and sequence analysis of the isolated tRNA show that the method is very specific. Individually labelled tRNA species can be used as probes for cloning tRNA genes.     

Purification of transfer RNA (m5U54)-methyltransferase from Escherichia coli. Association with RNA

tRNA (m5U54)-methyltransferase (EC 2.1.1.35) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to transfer ribonucleic acid (tRNA) and thereby forming 5-methyluridine (m5U, ribosylthymine) in position 54 of tRNA. This enzyme, which is involved in the biosynthesis of all tRNA chains in Escherichia coli, was purified 5800-fold. A hybrid plasmid carrying trmA, the structural gene for tRNA (m5U54)-methyltransferase was used to amplify genetically the production of this enzyme 40-fold. The purest fraction contained three polypeptides of 42 kDa, 41 kDa and 32 kDa and a heterogeneous 48-57-kDa RNA-protein complex. All the polypeptides seem to be related to the 42/41-kDa polypeptides previously identified as the tRNA (m5U54)-methyltransferase. RNA comprises about 50% (by mass) of the complex. The RNA seems not to be essential for the methylation activity, but may increase the activity of the enzyme. The amino acid composition is presented and the N-terminal sequence of the 42-kDa polypeptide was found to be: Met-Thr-Pro-Glu-His-Leu-Pro-Thr-Glu-Gln-Tyr-Glu-Ala-Gln-Leu-Ala-Glu-Lys- . The tRNA (m5U54)-methyltransferase has a pI of 4.7 and a pH optimum of 8.0. The enzyme does not require added cations but is stimulated by Mg2+. The apparent Km for tRNA and S-adenosyl-L-methionine are 80 nM and 17 microM, respectively.     

Nuclear amination catalyzed by fungal laccases: Comparison of laccase catalyzed amination with known chemical routes to aminoquinones

Laccases are able to initiate nuclear amination of p-hydroquinones with primary aromatic amines, resulting in the formation of the corresponding monoaminated and diaminated quinones. Two laccase catalyzed reactions are compared with established synthetic routes to aminoquinones, showing that formation of products from laccase catalyzed reaction is comparable with reaction using sodium iodate as oxidant. Advantages and disadvantages of laccase catalyzed amination are discussed. It is concluded that laccase catalysis is less suitable than sodium iodate oxidation for the amination of simple p-hydroquinones with simple amines.     

Effects of speciation on partitioning of iodine in aqueous biphasic systems and onto ABEC resins

Polyethylene glycol (PEG)-aqueous biphasic systems (ABS) and PEG-grafted aqueous biphasic extraction chromatographic (ABEC) resins have been shown to remove inorganic species from environmental and nuclear wastes. The partitioning behavior of several iodide species (iodide, iodine, triiodide, iodate, and 4-iodo-2,6-dimethylphenol (I-DMP)) have been studied for PEG (MW 2000)-salt systems and ABEC resins. Iodide partitioning to PEG-rich phases or onto ABEC resins can be enhanced by derivatization with 2,6-dimethylphenol to form 4-iodo-2,6-dimethylphenol or by addition of I(2) to form triiodide. Conversely, iodide partitioning to the PEG-rich phase or onto ABEC resins is reduced by oxidation of iodide to IO(3)(-). Partitioning studies of iodide, iodate, and iodine in a PEG-ABS are compared to results using ABEC resins.     

Nutrient speciation and hydrography in two anchialine caves in Croatia: tools to understand iodine speciation

Despite iodine being one of the most abundant of the minor elements in oxic seawater, the principal processes controlling its interconversion from iodate to iodide and vice versa, are still either elusive or largely unknown. The two major hypotheses for iodate reduction involve either phytoplankton growth in primary production, or bacteria during regeneration. An earlier study intended to exploit the unusual nature of anchialine environments revealed that iodide is oxidised to iodate in the bottom of such caves, whereas reduction of iodate occurs in the shallower parts of the water column. This investigation was made on the hypothesis that study of the nitrogen and phosphorus nutrient systems within the caves might offer a bridge between the iodine chemistry and the marine bacteria which are assumed to be the agent of change of the iodine in the caves. Accordingly, the hydrography, the nutrient chemistry, and some further iodine studies were made of two anchialine caves on the east coast of the Adriatic Sea in Croatia. Iodate and iodide were determined by differential pulse voltammetry and cathodic stripping square-wave voltammetry, respectively. Total iodine was determined indirectly, as iodate, after oxidation of reduced iodine species with UV irradiation and strong chemical oxidants. Nutrient concentrations were measured by spectrophotometry. Nutrient profiles within the well stratified water columns indicate a relatively short-lived surface source of nitrate and phosphate to the caves, with a more conventional, mid-water, nutrient regeneration system. The latter involves nitrite and ammonium at the bottom of the halocline, suggestive of both autotrophic and heterotrophic microbial activity. High iodate/low iodide deep water, and conservative behaviour of total inorganic iodine were confirmed in both systems. Iodate is reduced to iodide in the hypoxic region where nutrient regeneration occurs. The concentrations of organic iodine were surprisingly high in both systems, generally increasing toward the surface, where it comprised almost 80% of total iodine. As with alkalinity and silica, the results suggest that this refractive iodine component is liberated during dissolution of the surrounding karst rock. A major, natural flushing of one of the caves with fresh water was confirmed, showing that the cave systems offer the opportunity to re-start investigations periodically.     

Structural effects of hypermodified nucleosides in the Escherichia coli and human tRNALys anticodon loop: the effect of nucleosides s2U, mcm5U, mcm5s2U, mnm5s2U, t6A, and ms2t6A

Previous nuclear magnetic resonance (NMR) studies of unmodified and pseudouridine39-modified tRNA(Lys) anticodon stem loops (ASLs) show that significant structural rearrangements must occur to attain a canonical anticodon loop conformation. The Escherichia coli tRNA(Lys) modifications mnm(5)s(2)U34 and t(6)A37 have indeed been shown to remodel the anticodon loop, although significant dynamic flexibility remains within the weakly stacked U35 and U36 anticodon residues. The present study examines the individual effects of mnm(5)s(2)U34, s(2)U34, t(6)A37, and Mg(2+) on tRNA(Lys) ASLs to decipher how the E. coli modifications accomplish the noncanonical to canonical structural transition. We also investigated the effects of the corresponding human tRNA(Lys,3) versions of the E. coli modifications, using NMR to analyze tRNA ASLs containing the nucleosides mcm(5)U34, mcm(5)s(2)U34, and ms(2)t(6)A37. The human wobble modification has a less dramatic loop remodeling effect, presumably because of the absence of a positive charge on the mcm(5) side chain. Nonspecific magnesium effects appear to play an important role in promoting anticodon stacking. Paradoxically, both t(6)A37 and ms(2)t(6)A37 actually decrease anticodon stacking compared to A37 by promoting U36 bulging. Rather than stack with U36, the t(6)A37 nucleotide in the free tRNAs is prepositioned to form a cross-strand stack with the first codon nucleotide as seen in the recent crystal structures of tRNA(Lys) ASLs bound to the 30S ribosomal subunit. Wobble modifications, t(6)A37, and magnesium each make unique contributions toward promoting canonical tRNA structure in the fundamentally dynamic tRNA(Lys)(UUU) anticodon.     

The T-arm of tRNA is a substrate for tRNA (m5U54)-methyltransferase

Fragments of Escherichia coli FUra-tRNA(1Val) as small as 15 nucleotides form covalent complexes with tRNA (m5U54)-methyltransferase (RUMT). The sequence essential for binding includes position 52 of the T-stem and the T-loop and extends toward the 3' acceptor end of FUra-tRNA. The in vitro synthesized 17mer T-arm of E. coli tRNA(1Val), composed of the seven-base T-loop and 5-base-pair stem, is a good substrate for RUMT. The Km is decreased 5-fold and kcat is decreased 2-fold compared to the entire tRNA. The T-arm structure could be further reduced to an 11mer containing the loop and two base pairs and still retain activity; the Km was similar to that of the 17mer T-arm, whereas kcat was decreased an additional 20-fold. The data indicate that the primary specificity determinants for the RUMT-tRNA interaction are contained within the primary and secondary structure of the T-arm of tRNA.     

Status of tRNA charging, trinucleotide acceptor sequence and tRNA nucleotidyltransferase activity in the human placenta.

Samples of tRNA isolated from the cell sap of full-term human placenta were found to have a low capacity for accepting amino acids in the presence of partially purified synthetase preparations made from placental or rat liver cell sap. Gel electrophoresis of placental tRNA showed that part of this could be accounted for by gross degradation. The proportion of chargeable tRNA carrying amino acids was estimated by periodate oxidation followed by stripping and then charging with labeled amino acids. Only 50% of chargeable placental tRNA was in the charged state when isolated, whereas 87% of freshly isolated rat liver tRNA was found to be charged with amino acids. A fraction from placental cell sap was shown to have tRNA nucleotidyltransferase activity. When placental tRNA was incubated with this fraction and [3H]ATP or [3H]CTP, ATP was incorporated into about 12% of the tRNA molecules and CTP into 5-7%. When rat liver tRNA was used in place of placental tRNA, [3H]ATP was incorporated into less than 5% of the tRNA molecules. By using snake-venom diesterase over short periods of incubation, it was confirmed that the ATP had been incorporated terminally as AMP into the placental tRNA. These observations show that, in contrast to rat liver tRNA, tRNA prepared from human placenta is poorly charged with amino acids, many of the molecules lack the acceptor trinucleotide and there is extensive degradation beyond this stage.     

Stringent regulation of the synthesis of a transfer ribonucleic acid biosynthetic enzyme: transfer ribonucleic acid(m5U)methyltransferase from Escherichia coli.

This paper describes the regulation of a transfer ribonucleic acid (tRNA) biosynthetic enzyme, the tRNA(m5U)methyltransferase (EC 2.1.1.35). This enzyme catalyzes the formation of 5-methyluridine (m5U, ribothymidine) in all tRNA chains of Escherichia coli. Partial deprivation of charged tRNAVal can be imposed by shifting strains carrying a temperature-sensitive valyl-tRNA ligase from a permissive to a semipermissive temperature. By using two such strains differing only in the allelic state of the relA gene, it was possible to show the tRNA(m5U)methyltransferase to be stringently regulated. Upon partial deprivation of charged tRNAVal, the differential rate of tRNA(m5U)methyltransferase synthesis was found to decrease in a strain with stringent RNA control (relA+), whereas it increased in the strain carrying the relA allele. This increase of accumulation of tRNA(m5U)methyltransferase activity required protein synthesis. Thus, when tRNA is partially uncharged in the cell, the relA gene product influences the expression of tRNA(m5U)methyltransferase gene.     

Determination of the degree of in vivo tRNA aminoacylation in yeast cells

A method is described to harvest yeast cells, extract tRNAs having a good biological activity, and measure the degree of in vivo tRNA aminoacylation. This measuremet is based on a determination of the percentage of tRNA which is resistant to periodate oxidation; control experiments have been performed to check that this treatment inactivates uncharged tRNA molecules, but does not affect aminoacylated ones, and allows therefore an accurate determination of the percentage of aminoacylated tRNA molecules.     

An investigation into the effects of iodide and iodate on plant growth

Summary An experiment was carried out in water cultures to compare the effect of iodide and iodate on plant growth. The experimental crop was oats which is known to be very sensitive to an overdose of iodine.Iodide led to a more pronounced growth depression than did iodate. The rate of uptake of iodine from iodide was more than double that from iodate. There appeared to be a fairly quantitative relation between the iodine content of the oat roots and their growth with high supplies of KI and KIO₃. This indicates that iodide and iodate, after being absorbed by the plant, have about the same effect on growth and that differences in effect during the initial stages of growth can be largely ascribed to differences in iodine contents.     

Covalent adducts between tRNA (m5U54)-methyltransferase and RNA substrates.

The interaction of tRNA (m5U54)-methyltransferase (RUMT) with in vitro synthesized unmodified tRNA and a 17-base oligoribonucleotide analog of the T-arm of tRNA in the absence of AdoMet has been investigated. Binary complexes are formed which are isolable on nitrocellulose filters and are composed of noncovalent and covalent complexes in nearly equal amounts. The covalent RUMT-RNA complexes are stable to SDS-PAGE and migrate slower than free enzyme or RNA. Kinetic and thermodynamic constants involved in formation and disruption of noncovalent and covalent binary complexes have been determined and interpreted in the context of steady-state kinetic parameters of the enzyme-catalyzed methylation and 5-H exchange of substrate. The results show that the isolable covalent complex is kinetically incompetent as an intermediate for methylation. Isotope trapping experiments show that when AdoMet is added to preformed binary complex, all bound tRNA is converted to methylated product; thus, the covalent complexes are chemically competent to form products. We have concluded that, after a reversible binary complex is formed, the catalytic thiol adds to the 6-carbon of the U54 of tRNA. The initial adduct leaves the reaction pathway to protonation at carbon 5; the latter can deprotonate and re-enter the pathway to form methylated product. It is speculated that covalent binary RUMT-RNA adducts may serve as depots of enzyme-tRNA complexes primed for methylation, or in unknown roles with RNAs other than tRNA.     

Sequence of a new tRNA(Leu)(U*AA) from brewer's yeast.

The nucleotide sequence of a new tRNA(Leu)(anticodon U*AA) from Saccharomyces cerevisiae which could recognize exclusively the UUA codon has been determined. Its primary structure is: pGGAGGGUUGm2GCac4CGAGDGmGDCDAAGGCm2(2)GGCAGACmUU*AAm1GA++ + psi CUGUUGGACGGUUGUCCGm5CGCGAGT psi CGm1A(orA)ACCUCGCAUCCUUCACCA. This tRNA has a large extraloop and contains 15 modified nucleotides. So far it is the third isoacceptor tRNA for leucine in yeast. It has 61% homology with tRNA(Leu)(anticodon m5CAA) and 63% homology with tRNA(Leu)(anticodon UAG), the two other known yeast tRNAs(Leu).     

The relationship of thermodynamic stability at a G x U recognition site to tRNA aminoacylation specificity

The G x U pair at the third position in the acceptor helix of Escherichia coli tRNA(Ala) is critical for aminoacylation. The features that allow G x U recognition are likely to include direct interaction of alanyl-tRNA synthetase with distinctive atomic groups and indirect recognition of the structural and stability information encoded in the sequence of G x U and its immediate context. The present work investigates the thermodynamic stability and acceptor activity for a comprehensive set of variant RNAs with substitutions of the G x U pair of E. coli tRNA(Ala). The four RNAs with Watson-Crick substitutions had a lower acceptor activity and a higher stability relative to the G x U RNA. On the other hand, the RNAs with mispair substitutions had a lower stability, but either a higher or a lower acceptor activity. Thus, the entire set of variant RNAs does not exhibit a correlation between thermodynamic stability of the free, unbound tRNA and its acceptor activity. The substantial acceptor activity of tRNAs with particular mispair substitutions may be explained by their ability to assume the conformational preferences of alanyl-tRNA synthetase. Moreover, the G x U pair may provide a point of deformability for the substrate tRNA to adapt to the enzyme's active site.     

Isotachophoresis for periodate oxidation analysis of a small amount of carbohydrate.

The present study demonstrates that formate, periodate, and iodate, in a reaction mixture for periodate oxidation of carbohydrates, are simultaneously and conveniently determined by isotachophoretic analysis. The operating condition of the electrophoretic method involves the addition of 0.3 vol of dioxane and 0.2% Triton X-100 to a deoxygenated leading electrolyte of 10 mm HCl buffered with 20 mm imidazole, pH 7.0. These additives are essential for complete resolution of formate and periodate in respective zones. It has been shown that the analyzable amounts of these products in the oxidation reaction are less than 5 nmol which corresponds to less than μg of carbohydrates. This value is about one-thousandth of that required for the conventional methods of periodate oxidation analysis.