Studies of odd bases in yeast mitochondrial tRNA: III. Characterization of the tRNA methylases associated with the mitochondria

Whereas m¹G, m²G, m₂²G, m⁷G, T, m¹A, m⁵C and Cm methylase activities were found in total cell enzyme of $\text{Saccharomyces cerevisiae}$ using under-methylated $\text{E. coli}$ tRNA and $\text{E. coli}$ B tRNA in reaction with or without Mg⁺⁺, only m¹G, m²G, m₂²G and T methylases occurred in mitochondria. Mitochondrial and cytoplasmic tRNA cannot be methylated by their homologous enzymes; only mitochondrial tRNA can be methylated in a heterologous reaction by total cell enzyme with formation of T, m⁵C, m¹A and low amounts of m²G and m₂²G.     

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.     

Purification and nucleoside composition of a Q* - containing aspartic acid tRNA from Drosophila.

One form of aspartic acid tRNA from $\text{Drosophila}$,$\text{melanogaster}$ (tRNA_2δ^Asp) is selectively bound to columns of Con A-Sepharose. Unlike the other Q-containing tRNAs of $\text{Drosophila}$, it therefore appears that tRNA^Asp contains the more highly modified nucleoside, Q¹ (mannose form) in its anticodon. This is further supported by the chromatographic insensitivity of tRNA_2δ^Asp to NaIO₄ treatment. Utilizing Con A-Sepharose chromatography, tRNA_2δ^Asp from $\text{Drosophila}$ was purified and its nucleoside composition determined by chemical tritium labelling. In addition to the major nucleosides, this tRNA contains rT, hU, m⁵C, ψ, and Q¹, but no other modified nucleosides. Its nucleoside composition is very similar to yeast tRNA^Asp.     

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.     

The stimulation of cerebral N2-methyl- and N2-2-dimethyl guanine-specific tRNA methyltransferases by methionine sulfoximine: an in vivo study.

The administration of a single convulsant dose or of multiple subconvulsant doses of L-methionine-dl-sulfoximine (MSO) to 18-day old rats results in a significant elevation of the specific activity of cerebral tRNA methyltransferases, as determined in an $\text{in vitro}$ assay, using heterologous or species-homologous tRNAs as substrates. The increase was detectable as early as 90 min after MSO and persisted throughout the entire 5–6 h preconvulsant period. The ¹⁴[C]-methyl tRNA was purified, and hydrolyzed to its constituent bases and their distribution was quantitated by high performance liquid chromatography. A marked increase in the formation of ¹⁴[C]-N²-methyl- and ¹⁴[C]-N₂²-dimethyl guanine was noted in the MSO-treated animals, demonstrating a specific stimulation by MSO $\text{in vivo}$ of the cerebral N²-methyl and/or N₂²-dimethyl guanine-specific tRNA methyltransferases.     

Separation of aminoacyl-transfer RNAs by polyacrylamide gel electrophoresis

A polyacrylamide gel electrophoresis system for separating $\text{E.}$$\text{coli}$ tRNAs and aminoacyl-tRNAs is described. The tRNA was separated into 6 discrete bands which contained varyin aamounts of tRNA and therefore varying numbers of tRNA species. In order to locate specific tRNAs, tRNA was charged with a ¹⁴C amino acid and the aminoacyl-tRNA was located by autoradiography. With several amino acids, 2 isoaccepting species were found. In total, 30 aminoacyl-tRNAs were located.     

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.     

N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA

In the biogenesis of adenovirus type 2 messenger RNAs, methylation occurs at the 5′ end (cap) and to internal adenosine residues to yield N⁶-methyl-adenosine (m⁶A) (Sommer et al., 1976; Moss &; Koczot, 1976; Wold et al., 1976). The kinetics of accumulation of ³H from methyl-labeled methionine and ¹⁴C from uridine into Ad-2²-specific RNA was measured late in Ad-2 infection. As reported previously (Nevins &; Darnell, 1978a), the rate of accumulation of [¹⁴C]uridine label in nuclear RNA was approximately four- to fivefold faster than in the cytoplasmic RNA, indicating a conservation of about 20% for the total RNA. The initial rates of [³H]methyl label in m⁶A in nuclear RNA and in the cytoplasmic RNA were approximately equal, suggesting a complete (or nearly complete) conservation of m⁶A.In accord with the accumulation kinetics, the ratio of ³H to ¹⁴C was higher in cytoplasmic RNA than in nuclear RNA that hybridized to equivalent regions of the Ad-2 DNA.A mathematical model was designed to evaluate the accumulation of methyl label in m⁶A, taking into consideration the three major parameters that affect the accumulation curves: equilibration of the S-adenosyl-methionine pool, the nuclear dwell time of sequences destined to be mRNA, and the cytoplasmic stability of mRNA. The half-time ($\text{t}\text{1}\text{2}$) for pool equilibration was determined experimentally to be 22 minutes and the nuclear dwell time and the mean life-time of cytoplasmic mRNA were estimated from ¹⁴C label to be about 30 and 70 minutes, respectively.The model gave an excellent fit to the data when the $\text{t}\text{1}\text{2}$ for pool equilibration time of 24 ± 2 minutes, a nuclear dwell time of 25 ± 10 minutes, and a mean cytoplasmic mRNA life-time of 75 ± 30 minutes were used to evaluate accumulation curves. Even when data from a restricted region of the genome, $\text{40.5–52.6}$, which encodes the main portion of at least five 3′ co-terminal mRNAs whose spliced junction with the tripartite leader sequence varies from $\text{38, 40, 43, 45}$, and $\text{48}$ was analyzed, it appeared that m⁶A was conserved.Finally, m⁶A was found to be added in a brief label (3.5 min) mainly to nuclear molecules that were longer than any cytoplasmic RNA. The conservation of m⁶A and its addition prior to splicing raise the possibility that internal methylations are involved, in the formation of mRNA.     

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.     

The uptake of (3H)dopamine by homogenates of rat corpus striatum: effects of cations

The uptake of [³H]dopamine was studied with a synaptosomal preparation of the corpus striatum. The accumulation of dopamine was found to be temperature-dependent and very rapid, but linear over time for at least 5 min. at 37°C with characteristics of saturable kinetics. The optimum concentrations for Na⁺ and K⁺ were 150–160 m$\text{M}$ and 2.5–4.8 m$\text{M}$, respectively, while uptake was progressively inhibited at concentrations of K⁺ greater than 5 m$\text{M}$. Rubidium was capable of substituting for potassium whereas cesium was a much less effective replacement. The uptake of DA was blocked by the antibiotics, valinomycin and gramicidin-D which bind K⁺ or both Na⁺ and K⁺, respectively, and thereby might interfere with the transport of cations across neuronal membranes. Similarly, ouabain which blocks the active transport of Na⁺ markedly antagonized the accumulation of DA into striatal homogenates. In contrast, tetrodotoxin which does not prevent the active transport of Na⁺, had no effect. Uptake appeared not to require Ca⁺⁺ and it was not inhibited by increasing total osmolarity to 400 mos$\text{M}$. In general, the cationic requirements for DA-uptake in striatal tissue and its responses to several inhibition of ionic transport, do not appear to be greatly different from those reported for NE with synaptosomes prepared from whole brain.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

Methylation of transfer RNA during transformation of human lymphocytes by phytohemagglutinin

Methylation of transfer RNA during phytohemagglutinin induced transformation of human lymphocytes was studied by labeling the tRNA $\text{in}\text{vivo}$ with (methyl-H³)-methionine and measuring the distribution of tritium in the methylated nucleotides. An alteration in the pattern of methylation occurred within hours after PHA-stimulation and this change was maintained through several cell generations. There was a 50 to 94% increase in the relative amount of methylated N²-methyl-guanine (1 to 3 hr) and a 40 to 59% decrease in the relative amount of 1-methyladenine (1 to 12 hr). Treatment of the stimulated cells with Actinomycin D prevented the subsequent methylation not tRNA. However, inhibition of protein synthesis by puromycin did not effect the early changes observed in the methylation of tRNA.     

The effects of environmental factors on growth and the chemical and biochemical composition of marine diatoms. I. Light and temperature effects

Temperature and light interact to modify the chemical and biochemical composition of a nitrogen-limited marine diatom, Thalassiosira allenii Takano, grown at a constant dilution rate in continuous culture and under a light:dark cycle.The percent of the total ¹⁴C incorporated into protein, polysaccharide and lipid, the N/C ratio and the C/cell varied with temperature in a markedly non-linear manner. The N/cell was negatively correlated to temperature. The Chl $\text{a}\text{C}$ ratio was positively correlated with temperature under saturating light and non-saturating light for temperatures > 25 °C, but was constant under non-saturating light conditions for temperatures < 25 °C.Productivity index (PI) was negatively correlated to temperature under saturating light conditions, but did not vary under low light. In each case, the variation in PI with temperature was governed by the variation in Chl $\text{a}\text{C}$.The dark carbon loss rate was exponentially related to temperature and independent of light. Variation in the percent of the total ¹⁴C incorporated into protein and polysaccharide, the $\text{N}\text{C}$ ratio and C/cell was primarily due to the effects of N-limitation < 20 °C and primarily due to the effects of temperature > 20 °C. Variation in N/cell was primarily due to the effects of temperature over the entire range of temperature studied. Variation in Chl $\text{a}\text{C}$ was caused by the interaction of temperature and light effects.In most cases, temperature and nutrient effects interacted to govern how a particular parameter varied with temperature while light affected the range of values over which the parameter varied.The percent of the total ¹⁴C incorporated into protein exhibited a significant linear relationship with $\text{N}\text{C}$.The dark carbon loss rate, $\text{N}\text{C}$ ratio and Chl $\text{a}\text{C}$ ratio data were used to test the applicability of a model for the physiological adaptation of unicellular algae. The model, with parameters derived from a non-linear least-squares fit of the dark carbon loss rate data, adequately described the $\text{N}\text{C}$ ratio between 15 and 25 °C at 290 and 137 μE · m^?2 · s^?1, but failed to describe the data at 28 °C and at 48 μE · m^?2 · s^?1. The Chl $\text{a}\text{C}$ ratio was adequately described by the model under all light and temperature conditions.     

Two forms of methionyl-transfer RNA synthetase from Mycobacterium smegmatis

Two methionyl-transfer RNA synthetases (A and B forms) have been isolated from $\text{Mycobacterium smegmatis}$. The homogeneous preparations of the enzymes showed 1500 fold increase in specific activity in aminoacylation of methionine specific tRNA. The A and B forms differed in their specificity of aminoacylation of tRNA_m^Met and tRNA_f^Met; enzyme B exhibited much higher specificity for tRNA_f^Met. The molecular activities of A and B enzymes for aminoacid and tRNA were identical. The turnover number for aminoacid was 27 fold greater than that for tRNA, while the Km values for tRNA were lower by a factor of 10⁶ as compared to the aminoacid. Both the enzymes catalysed ATP-pyrophosphate exchange reaction to the same extent.     

Effect of specific inhibitors on mitochondrial DNA replication in HeLa cells

The crystal structure of an orthorhombic form of 2′-0-methyl cytidine was determined from three dimensional X-ray diffraction data. The two molecules in each asymmetric unit have C2-$\text{endo}$ C3-$\text{exo}$ puckered furanose rings. This differs from the C3-$\text{endo}$ puckering observed for cytidine (1) and it may have some relevance to the kinks that appear at the two 2′-0-methylated nucleotides in the anticodon phosphate ester backbone of the phe tRNA structure (2). This work and other studies (3,4) show that the presence of a 2′-0-methyl group does not prevent the furanose moiety from adopting its most commonly observed configurations. 2′-0-methyl nucleotides make up a small percentage of the residues in HnRNA, rRNA, tRNA and mRNA and therefore their conformational nuances are of interest.     

One predominant 5′-undecanucleotide in adenovirus 2 late messenger RNAs

Oligonucleotides containing the 5′ termini of adenovirus 2 mRNA are selectively retained on columns of dihydroxyboryl cellulose. When total late adenovirus 2 mRNA was treated with RNAase T1, a single 5′ terminal oligonucleotide was isolated, although in several states of methylation. This oligonucleotide has the general structure m⁷GS^5′ppp^5′$\text{A}$mCmU(C₄,U₃)G. Since at least twelve individual species of mRNA must be present late after infection, this finding was unexpected and its significance is discussed.     

Methylation of late adenovirus 2 nuclear and messenger RNA.

Methylation of adenovirus 2 (Ad 2) late RNA was studied. RNA was double-labeled with [³H-methyl]-methionine and [¹⁴C]-uridine 15–20 h postinfection. Nuclear RNA (rRNA) and cytoplasmic RNA (mRNA) was extracted, and fractionated into polyA(+) and (?) molecules using poly(U)-Sepharose. Ad 2 specific RNA was purified by 2 cycles of hybridization to and elution from Ad 2 DNA immobilized on filters. The Ad 2 polyA(+) and (?) nRNA and mRNA fractions had the same ${}^3\text{H}{}^{14}\text{C}$ ratios, and were estimated to contain a minimum of 1.4 methylated nucleotides per 1000 bases. Viral RNA was digested with RNase T₂ and chromatographed on DEAE-Sephadex in 7 M urea at pH 7.6. All four Ad RNA fractions contained methylated constituents consistent with: (1) two classes of methylated “capped” 5′-termini with general structures m⁷ GpppN^mpNp and m⁷ GpppN^mpN^mpNp; (2) internal base methylations; (3) minor amounts of internal ribose 2′-0-methylations. Two classes of 5′-termini have previously been reported for animal cell mRNA, but not for mRNA from a variety of viruses. Internal methylations may be unique to RNA molecules transcribed in the nucleus, since they have not been found in RNA from cytoplasmic viruses. No gross differences were observed in the DEAE-Sephadex elution profiles of the methylated constituents of the four types of Ad 2 RNA. These results suggest that the majority of methylation events occur in the nucleus, and raise the possibility that Ad 2 methylated late nRNA may differ significantly from SV40 late nRNA (Lavi, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci. USA $\text{72}$, 2012–2016).     

Effect of phospholipid methylation on calcium transport and (Ca2+ + Mg2+)-ATPase activity in kidney cortex basolateral membranes

Basolateral membranes isolated from hog kidney cortex, enriched 12- to 15-fold in (Na⁺ + K⁺)-ATPase activity, were 80% oriented inside-out as determined by assay of oubain-sensitive (Na⁺ + K⁺)-ATPase activity before and after opening of the membrane vesicle preparation with a mixture of deoxycholate and EDTA. In these membrane preparations 80% of total phosphatidylethanolamine was accessible to trinitrophenylation by trinitrobenzenesulfonic acid at 4°C, while at 37°C all of phosphatidylethanolamine fraction was chemically modified. Phospholipase C treatment resulted in hydrolysis of 80% phosphatidylethanolamine, 40% phosphatidylcholine and 35% of phosphatidylserine. Sphingomyelinase treatment resulted in 20% hydrolysis of sphingomyelin, presumably derived from right-side-out oriented vesicles. Results indicate that phosphatidylethanolamine is oriented exclusively on the outer leaflet of the lipid bilayer of inside-out oriented vesicles. Methylation of phospholipids in basolateral membranes with $\text{S-}\text{adenosyl}\text{[methyl-}{}^3\text{H}\text{]}\text{methionine}$ resulted in the three successive methylation of ethanolamine moiety of phosphatidylethanolamine to phosphatidylcholine. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{S-}\text{adenosylmethionine}$ was 1·10^?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg²⁺ was without any effect between pH 5 and 10. Basolateral membranes incubated in the presence of methyl donor, $\text{S-}\text{adenosylmethionine}$, exhibited increased (12–15%) (Ca²⁺ + Mg²⁺)-ATPase activity and increased ATP-dependent uptake of calcium. ATP-dependent calcium uptake in these vesicles was insensitive to oligomycin and ouabain but was abolished completely by 50 μM vanadate. The increase in ATP-dependent calcium uptake was due to an increase in $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ and not due to a change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Ca²⁺. Preincubation of membranes with $\text{S-}\text{adenosylhomocysteine}$, a methyltransferase inhibitor, abolished the stimulatory effect of phospholipid methylation on calcium uptake. Phospholipid methylation at both low and high pH did not result in a change in bulk membrane fluidity as determined by the fluorescence polarization of diphenylhexatriene. These results suggest that phospholipid methylation may regulate transepithelial calcium flux in vivo.     

Aminoacyl transfer from phenylalanyl-tRNA microinjected into Xenopus laevis oocytes.

$\text{Xenopus laevis}$ oocytes were injected with [¹⁴C] phe-tRNA and the fate of the aminoacyl moiety was studied. The radioactive phenylalanine is gradually hydrolized off the tRNA once inside the cell. The rate of deacylation of the tRNA is not affected by inhibition of cellular protein synthesis by puromycin or cycloheximide. Part of the radioactive amino acid that leaves the tRNA (30 to 65%) is transferred directly into the oocyte nascent proteins as evidenced by the fact that its incorporation into proteins is not reduced by coinjection with a large excess of [¹²C] phenylalanine. Aminoacyl transfer from injected phe-tRNA into proteins is inhibited by puromycin and cycloheximide.     

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.