Studies on the ribothymidine content of specific rat and human tRNAs: a postulated role for 5-methyl cytosine in the regulation of ribothymidine biosynthesis.

Total mammalian tRNAs contain on the average less than one mole of ribothymidine per mole of tRNA. Mammalian tRNAs can be grouped into at least four classes, depending upon their ribothymidine content at position 23 from the 3′ terminus. Class A contains tRNA in which a nucleoside other than uridine replaces ribothymidine (tRNA_i^Met); Class B contains tRNA in which one mole of a modified uridine (rT, ψ, or 2′-O-methylribothymidine) is found per mole of tRNA (tRNA^Ser, tRNA^Trp, and tRNA^Lys, respectively). Class C contains tRNA in which there is a partial conversion of uridine to ribothymidine (tRNA^Phe, tRNA₁^Gly, tRNA₂^Gly); Class D contains tRNA which totally lacks ribothymidine (tRNA^Val). Only those tRNAs in Class C are acceptable substrates for $\text{E.}\text{coli}$ uridine methylase, under the conditions used in these studies. These observations cannot be adequately explained solely on the basis of the presence or absence of a specific “universal” nucleoside other than U or rT at position 23 from the 3′ terminus. However, correlations can be made between the ribothymidine and 5-methylcytosine content of eucaryotic tRNA. We postulate that the presence of one or more 5-methylcytosines in and adjacent to loop III (minor loop) in individual tRNAs act to regulate the amount of ribothymidine formed by uridine methylase. Several experiments are proposed as tests for this hypothesis.     

Complex formation between transfer RNAs with complementary anticodons: use of matrix bound tRNA

It is shown that yeast tRNA^Phe, chemically coupled by its oxidized 3′CpCpA end behaves exactly as free tRNA^Phe in its ability to form a specific complex with $\text{E. coli}$ tRNA₂^Glu having a complementary anticodon. The results support models of tRNA in which the 3′CpCpA_OH end and the anticodon are not closely associated in the tertiary structure, and provide a convenient tool of general use to characterize others pairs of tRNA having complementary anticodons, as well as for highly selective purification of certain tRNA species.     

Studies of odd bases in yeast mitochondrial tRNA: II. Characterization of rare nucleosides

The nucleoside composition of tRNA from highly purified yeast mitochondria shows the presence of T, ψ, hU, m¹G, m²G, m₂²G, I and t⁶A whereas neither m⁷G, m⁵C, m³C, m¹A, i⁶A and Y nor O′-methylated nucleosides (which are common in yeast cytoplasmic tRNA) were found. The G+C content is very low (35%). The overall methylation content is 2.7% which is about half the content of yeast cytoplasmic tRNA but similar to that of $\text{E. coli}$ tRNA. Some rare nucleosides however which are found in $\text{E. coli}$ (s⁴U, acp³U, m²A, m⁶A, ms²i⁶A, Q) were not found in yeast mitochondrial tRNA.     

A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon

Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA^Asp QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA^Asp isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNA^Asp anticodon stem and the tRNA^Glu amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA^Asp synthetases, is conserved among prokaryotes.     

NMR study of the modified base resonances of tRNA tyr- coli

220MHz NMR spectra at 28° show several resolved resonances in the high field region for D₂O solutions of tyrosine specific tRNA from E. coli. These resonances are tentatively identified as arising from protons of the modified nucleoside, 2-methylthio-N⁶-(Δ²-isopentenyl)-adenosine and from the modified guanosine of unknown structure in the “wobble position” of the anti codon loop. Assignment of resonances was aided by comparison with spectra of tRNA_su⁺III^tyr, Form II, whose sequence is closely homologous to tRNA_coli^tyr, except for changes in some modified bases. Line widths of resolved resonances indicate that, at 28°, the methyl groups of modified nucleosides are not completely restricted in their motion relative to the overall motion of the macromolecule.     

Selective changes in methionine tRNA species during development of the posterior silkglands of Bombyx mori.

Transfer RNA with methionine acceptor activity isolated from two distinct physiological stages of the developing posterior silkgland of the silkworm, $\text{Bombyx mori}$, was examined. The tRNA from both stages could be fractionated on benzoylated DEAE-cellulose colum into two iso-accepting species, tRNA₁^Met and tRNA₂^Met. The molar quantity per gland of tRNA₁^Met species, which was also formylatable with the $\text{E. coli}$ enzymes, increased twelve-fold as the gland differentiates to produce a large amount of a single protein, silk-fibroin. Since methionine is not a part of silk-fibroin, the preferential increase in tRNA₁^Met content would reflect the increased biological activity and the rapid rate of protein synthesis during the terminal differentiation of posterior silkgland.     

Sequences of three transfer RNAs from mosquito mitochondria

The sequences of three transfer RNAs from mosquito cell mitochondria, tRNA_UCG^Arg, tRNA_GUC^Asp, and tRNA_GAU^Ile, determined using a combination of rapid ladder and fingerprinting procedures are reported. These were compared with hamster mitochondrial tRNA_UCG^Arg and tRNA_GUC^Asp determined similarly, and a bovine mitochondrial tRNA_GAU^Ile determined using a somewhat different approach. The primary sequences of the mosquito tRNAs were 35 to 65% homologous to the corresponding mammalian mitochondrial species, and bore little homology to “conventional” (bacterial or eucaryotic cytoplasmic) tRNA. The modification status of the mosquito mitochondrial tRNAs resembled that of mammalian mitochondrial tRNA. The results contribute to the generalization that metazoan mitochondrial tRNA constitutes a distinctive, albeit loosely structured, phylogenetic group.     

The localization of tRNA 5 Asn,tRNAHis, and tRNAAla genes from Drosophila melanogaster by in situ hybridization to polytene salivary gland chromosomes

Transfer RNA _5; ^Asn , tRNA _; ^His , and tRNA^Ala were isolated from Drosophila melanogaster by means of Sepharose 4B chromatography and 2-dimensional polyacrylamide gel electrophoresis. The tRNAs were iodinated in vitro with Na¹²⁵I and hybridized in situ to salivary gland chromosomes from Drosophila. Subsequent autoradiography allowed the localization of the genes for tRNA _5; ^Asn in the regions 42A, 59F, 60C, and 84F; for tRNA^His in the regions 48F and 56E; and for tRNA^Ala in the regions 63A and 90C. From these and our previous results it can be concluded that the genes for the Q-base containing tRNAs (tRNA^Asn, tRNA^Asp, and tRNA^His, are not clustered in the Drosophila melanogaster genome.     

Elongator complex influences telomeric gene silencing and DNA damage response by its role in wobble uridine tRNA modification

Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm⁵U₃₄), 5-methoxycarbonylmethyluridine (mcm⁵U₃₄), and 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U₃₄) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA^Lys _{s² UUU}, tRNA^Gln _{s² UUG}, and tRNA^Glu _{s² UUC}, which in a wild-type background contain the mcm⁵s²U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U₃₄. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm⁵s²U nucleoside in tRNA^Lys _mcm⁵s²UUU, tRNA^Gln _mcm⁵s²UUG, and tRNA^Glu _mcm⁵s²UUC. These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U₃₄ are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.     

Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5′ end of the molecule. This group of tRNAs includes E. coli tRNA^Nfmet, tRNA^Ala₁, tRNA^Leu₁, or ^Leu₂, and tRNA^Ser₃ (Group 1). In each case N²-methylguanine and N²,N²-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N²-guanine methyltransferase II ($\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine:tRNA}$ (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50°C for 10 min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preperations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N²-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

Anomalies in 5'-end [32P] labelling of transfer RNA and partial sequence of a tRNAGlu from Scenedesmus obliquus

A chloroplast tRNA_m^Met species from $\text{Scenedesmus}\text{obliquus}$ is very poorly 5′-end [${}^{32}\text{P}$] labelled using [γ-³²P]ATP and T4 polynucleotide kinase. In sequencing the tRNA using standard 5′-labelled methods a very minor contaminating tRNA is preferentially labelled. The partial tRNA sequence determined by this method has an anticodon (CUC) for tRNA^Glu.     

Reversible modification of arginine residues with glyoxal

Animal serum contains an activity, designated Q-factor, which effects an increase in nucleoside Q-containing tRNA in tissue culture. The appearance of Q-positive tRNA^Asp in the L-M cell line cultivated serum-free has been used as an assay to partially characterize Q-factor from fetal bovine serum and to determine that bovine amniotic fluid contains 100 fold more Q-factor than does fetal bovine serum. Q-factor is dialyzable, 500 molecular weight or less, and binds tightly to activated charcoal and dextran. Using Q-factor, evidence is presented that the Q-negative tRNA^Asp species are precursors of the Q-positive species.     

Mechanism of suppression in Drosophila: II. Enzymatic discrimination of wild-type and suppressor tyrosine transfer RNA

Previous studies had shown that two principle forms of tyrosine transfer RNA of Drosophila melanogaster were present in wild-type adult flies but that the second form was virtually absent in a suppressor mutant, su(s)². Current results are at variance with the previous ones, in that the suppressor mutant has significant amounts of the second form of tRNA^Tyr. A second chromatography system for separating these forms of tRNA^Tyr is described, RPC-5, and is compared to the system used previously, RPC-2. Both systems indicate that wild-type flies contain the two forms of tRNA^Tyr in a ratio of $\text{40}\text{60}$, the suppressor mutant in a ratio of $\text{60}\text{40}$. The difference between current and previous results can be attributed to the procedures used in the preparation of the enzyme that is used as a source of tyrosyl-tRNA ligase. The enzyme activity can be separated into two fractions on DEAE-cellulose chromatography. With suppressor tRNA as substrate, one enzyme fraction charges both forms of tRNA^Tyr but the second enzyme fraction charges the first form preferentially or nearly exclusively in some cases, as was seen in the previous experiments. With wild-type tRNA as substrate both enzyme fractions charge both forms of tRNA^Tyr. Storage results in the loss of the enzyme's ability to discriminate against the second form of tRNA^Tyr from the suppressor mutant, while the enzymatic activity is retained. We postulate that the su(s)⁺ locus produces an enzyme that modifies the second isoacceptor of tRNA^Tyr and that, when such modification fails to occur (as in the su(s)² mutant), the tRNA is unable to accept tyrosine from one form of tyrosyl-tRNA ligase. How the discrimination against the second isoacceptor by the ligase may be important metabolically is not apparent.     

Recognition of altered E. coli formylmethionine transfer RNA by bacterial T factor

Treatment of $\text{E.}\text{coli}$ formylmethionine tRNA with sodium bisulfite produces six C → U base changes in the tRNA structure. Four of these modifications have no effect on the ability of tRNA_f^Met to be aminoacylated or formylated. Prior to bisulfite treatment, Met-tRNA_f^Met is not able to form a ternary complex with bacterial T factor and GTP, as measured by Sephadex G-50 gel filtration. After bisulfite treatment, a large portion of the modified tRNA is bound as T-GTP-Met-tRNA_f^Met. Formylation of bisulfite-modified Met-tRNA_f^Met completely eliminates T factor binding. Unmodified tRNA_f^Met is unique among the tRNAs sequenced to date in having a non-hydrogen-bonded base at the 5′ terminus. Bisulfite-catalyzed conversion of this unpaired C₁ to U₁ results in formation of a normal U₁-A₇₃ base pair at the end of the acceptor stem. It is likely that this structural alteration is responsible for the recognition of bisulfite-modified Met-tRNA_f^Met by T factor.     

Separation and comparison of primary structures of three formylmethionine tRNAs from E. coli K-12 MO

Three chromatographically distinct tRNAs^fMet from $\text{E. coli}$ K-12 MO were separated by reversed-phase chromatography and designated tRNA_A^fMet, tRNA_B^fMet, and tRNA₃^fMet. The tRNA_A^fMet corresponds to the published sequence for tRNA^fMet ($\text{E. coli}$). The tRNA_B^fMet differs from tRNA_A^fMet in that the 4-thiouridine in nucleotide position 8 has interacted with cytidine in position 13 to form a cross-linked product. The tRNA₃^fMet differs from tRNA_A^fMet in that 7-methyl-guanosine (in position 47) has been replaced by adenosine.     

Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding

Transfer RNA structure involves complex folding interactions of the TΨC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TΨC domain, rT₅₄, Ψ₅₅ and m⁵C₄₉, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA^Phe T-half molecules (nucleosides 40–72) with the corresponding unmodified D-half molecule (nucleosides 1–30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg²⁺ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K_ds (rT₅₄ = 6 ± 2, m⁵C₄₉ = 11 ± 2, Ψ₅₅ = 14 ± 5, and rT₅₄,Ψ₅₅ = 11 ± 3 µM) significantly lower than that of the unmodified T-half (40 ± 10 µM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA^Phe and native yeast tRNA^Phe, as determined by lead cleavage patterns at U₁₇ and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA’s TΨC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.     

Modified nucleosides and the chromatographic and aminoacylation behavior of tRNAIle from Escherichia coli C6

Transfer RNA from Escherichia coli C6, a Met⁻, Cys⁻, relA⁻ mutant, was previously shown to contain an altered tRNA^Ile which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676–7683). We now report the purification of this altered tRNA^Ile and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNA^Ile purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNA^Ile (from cysteine-starved cells) was found to have a 5-fold increased V_max in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl ∼ AMP · Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNA^Ile bound more efficiently to its synthetase compared to normal tRNA^Ile. Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNA^Ile contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNA^Ile contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNA^Ile without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNA^Ile has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNA^Ile from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNA^Ile for aminoacylation is discussed.     

Interaction of manganese with fragments, complementary fragment recombinations, and whole molecules of yeast phenylalanine specific transfer RNA

Binding of Mn²⁺ to the whole molecule, fragments and complementary fragment recombinations of yeast tRNA^Phe, and to synthetic polynucleotides was studied by equilibrium dialysis. The comparison of the binding patterns of the fragments, fragment recombinations and synthetic polynucleotides with that of intact tRNA^Phe permits reasonable conclusions concerning the nature and location of the various classes of sites on tRNA^Phe. Binding of Mn²⁺ to intact tRNA^Phe consists of a co-operative and a non-co-operative phase. There are about 17 “strong” sites and several “weak” ones. Five of the 17 strong sites are associated with the co-operative phase. This phase is completely lacking in the binding of Mn²⁺ to tRNA^Phe fragments (5′-$\text{1}\text{2}$, 3′-$\text{1}\text{2}$, 5′-$\text{3}\text{5}$, 3′-$\text{2}\text{5}$), poly-(A):poly(U) and poly(I):poly(C) helices, and single stranded poly(A) and poly(U). This argues that the co-operative sites arise from the tRNA tertiary structure. This conclusion is further strengthened by the observation that cooperativity is present in a tRNA^Phe molecule which has been split in the anticodon loop, but it is absent in one which has been split in the extra loop. It is in the vicinity of the latter loop, but not the former, that tertiary interactions are seen in the crystal structure. The remaining 12 strong sites are “independent” and appear to be associated with cloverleaf helical sections.     

Primary structure of E. coli alanine transfer RNA: relation to the yeast phenylalanyl tRNA synthetase recognition site

Nucleotide sequence comparison of tRNAs aminoacylated by yeast phenylalanyl tRNA synthetase (PRS) have lead to the proposal that the specific nucleotides of the dihydrouridine (diHU) stem region and adenosine at the fourth position from the 3′ end are involved in the PRS recognition site. Kinetic analysis and enzymatic methylation have shown that the size of the diHU loop and the methylation of guanine at position 10 from the 5′ end both directly affect the PRS aminoacylation kinetics. $\text{E. coli}$ tRNA₁^A1a, which is aminoacylated by PRS, should therefore have 1- the specific nucleotides of the diHU stem region and, 2- adenosine at position 4 from the 3′ end. The PRS aminoacylation kinetics of this tRNA indicates that this molecule 3- has a diHU loop of 8 nucleotides and 4- has an unmethylated guanine at position 10 from the 5′ end. We report here the complete sequence of $\text{E. coli}$ tRNA₁^A1a and confirmation of each of these four predictions.