Cell growth and tRNA4 synthesis in mouse 3T3 cells

The RPC-5 chromatographic profiles of lys-tRNA were analyzed during the growth of 3T3 cells in culture. An inverse relationship was seen between tRNA₂^lys and tRNA₄^lys which was markedly influenced by medium changes. This interchange of tRNA₂^lys and tRNA₄^lys could be controlled by altering the levels of serum in the medium, or more precisely by altering the serum to cell ratio. A different change in lys-tRNA distribution was seen when the cells reached confluency. The amounts of tRNA₂^lys, tRNA₃^lys and tRNA₄^lys all decreased with a corresponding increase in either tRNA₅^lys or tRNA₆^lys. An identical change in lys-tRNA could be produced by shifting sparse cells into a medium containing 10% calf plasma instead of 10% serum. Both tRNA^lys profiles and cell growth were returned to normal when the cells were returned to medium with 10% fetal calf serum (FCS) or 10% calf plasma and fibroblast growth factor (FGF). A third alteration in tRNA^lys profiles was seen by the addition of cAMP to the cultures. A decrease in tRNA₅^lys and a corresponding increase in tRNA₆^lys was seen upon the addition of 10⁻³ M db-cAMP and was accentuated by the simultaneous addition of 10⁻³ M methyl isobutylxanthine.These data are consistent with an ordered sequence of tRNA^lys modification involving tRNA₂^lys, tRNA₃^lys, tRNA₄^lys, tRNA_5B^lys and tRNA₆^lys. Several of the factors which control proliferation appear to control the activity of different tRNA-modifying enzymes in this tRNA^lys pathway thereby controlling the levels of tRNA₄^lys, a tRNA previously shown to correlate directly with the proliferative rate of cells.     

Lys-tRNA4 levels and cell division in mouse 3T3 cells

tRNA₄^lys is an isoaccepting tRNA^lys which has been proposed as a necessary requirement for cell division in mammalian cells. We have measured the levels of this tRNA^lys during the growth cycle of mouse 3T3 fibroblasts. High levels of tRNA₄^lys were seen throughout exponential growth. However, a marked decrease in tRNA₄^lys occurred 24 h before the cells became confluent. This decrease was observed in three different 3T3 cell lines, but was not seen in a transformed 3T3 cell line. Trypsinization and replating of contact-inhibited cells returned tRNA₄^lys to the levels characteristic of exponential cells. Data from these and other cell lines show a direct relationship between the levels of tRNA₄^lys and the growth rate of cells in culture.     

Lys-tRNA4 and cell division : Changes in lys-tRNA during the growth of mouse L cells

The presence of one isoaccepting tRNA species, lys-tRNA₄, has been correlated with the ability of mammalian cells to carry out cell division. The levels of this tRNA were measured during the growth of mouse L cells in culture. RPC-5 profiles of the lys-tRNA population of mouse L cells were determined at 9 points during the growth of mouse L cells in culture. The level of lys-tRNA₄ was constant at 13–14% of the total lys-tRNA population during log growth, increased to 18% when the cells became confluent and then returned to 13–14% when the cells were held at confluency. These results are in contrast to other cell lines where lys-tRNA₄ decreased at confluency. Confluent cultures of mouse L cells retained full proliferative potential since the addition of medium containing 30% serum led to renewed exponential growth. Another lys-tRNA species, lys-tRNA_5A, was present in variable amounts depending upon the nutritive state of the cells. The appearance of lys-tRNA_5A correlated with a decrease in lys-tRNA₅. These data suggest that lys-tRNA_5A is a modified lys-tRNA₅ which appears when the medium becomes depleted.     

Role of mitogen-induced calcium influx in the control of the cell cycle in Balb-c 3T3 fibroblasts

The role of mitogen-activated calcium influx from the extracellular medium in the control of cell proliferation was studied in Balb-c 3T3 fibroblasts. Stimulation of serum-deprived, quiescent cells with 10% foetal calf serum (FCS) induced a long-lasting (up to 70 min elevation of intracellular free calcium concentration ([Ca²⁺]_i). Both the sustained [Ca ²⁺]_i increase and the related inward current, described in a previous paper [Lovisolo D. Munaron L. Baccino FM. Bonelli G. (1992) Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts. Biochim. Biophys. Acta, 1104, 73–82], could be abolished either by chelation of extracellular calcium with EGTA or by SKF 96365, an imidazole derivative that can block receptor-activated calcium channels. The effect of the abolition of these ionic signals on FCS-induced proliferation was investigated by adding either EGTA or SK&F 96365 to the culture medium during the first hours of stimulation of quiescent cells with 10% FCS. As measured after 24 h, a 22% inhibition of growth was observed when SK&F 96365 was added for the first hour, and stronger inhibitions, up to 56%, were obtained by adding the blocker for the first 2 or 4 h. Similar effects were observed with addition of 3 mM EGTA, though the inhibition was less marked for the 4 h treatment. By contrast, incubation with either substance in the next 4 h of serum stimulation did not influence cell growth, except for a slight inhibition observed when SKF 96365 was applied from the 4^th to the 8^th hour. The reduction in growth resulting from the abolition of the early calcium influx was paralleled by an accumulation of cells in the G₂/M phase. Both growth inhibition and G₂/M accumulation were reversible, since after further 24 h in 10% FCS cells had fully recovered the exponential growth. These data indicate that the early calcium influx seen in response to mitogen stimulation develops on a timescale long enough to play a significant role in cell cycle progression, and that its block in the early G1 phase can lead to a reduction of proliferation by arresting cells in later stages of the cycle.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing aminopterin

When seeded in small numbers in medium containing 10^?6M aminopterin and fetal calf serum, V₇₉ Chinese hamster cells required dialyzable components from the serum for growth. However, the cells grew in medium containing 10^?6M aminopterin and dialyzed serum, provided that the medium was supplemented with 10^?5M hypoxanthine and sufficient 5·10^?6M) thymidine. A growth-inhibitory property of some batches of dialyzed serum was abolished on heating the serum for 30 min at 56°. Three lines of V₇₉ cells which lacked detectable hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity were seleccted in medium containing 8-azaguanine (8-AzG). In two of these, no spontaneous reversion to the HGPRT⁺ phenotype was detectable, and these cells did not cooperate metabolically with HGPRT⁺ cells to prevent the growth of the latter in HAT medium. One of the HGPRT^? lines showed a high rate of spontaneous reversion (118/10⁵ cells) in medium containing undialyzed serum. However, in medium containing dialyzed serum the spontaneous reversion rate fell to $\text{4}\text{10}{}^5\text{cells}$, suggesting that the revertants arising in medium containing undialyzed serum were biochemically heterogeneous.     

Relationship among the genes, enzymes, and intermediates of the biosynthetic pathway of lysine in Saccharomyces

Summary Genetic lesions of 10 of the lysine loci in Saccharomyces have been provisionally correlated with the various enzymatic steps of the homocitric acid pathway for the biosynthesis of lysine. The characterization of mutational blocks in different mutants was made on the basis of the accumulation of appropriate intermediates, lack of enzymatic activity, and feeding experiments. Genetic lesions in mutants lys ₄, lys₇, lys₈, and lys ₁₂ correspond to the biochemical steps between homocitric acid and -amino-adipic acid. Similarly the genetic lesions in lys ₁, lys₂, lys₅, lys₉, lys₁₃, and lys ₁₄ correspond to the intermediate steps between -aminoadipic acid and lysine. Lysine-genes are not clustered, instead are mapped on different chromosomes of Saccharomyces. Some of the intermediate steps in the biosynthesis of lysine correspond to two or more unlinked genes.Supported by NSF Grant GB 28558X, Lilly Research Foundation, and Faculty Research Committee, Miami University.     

UDP-galactose inhibition of BALB/3T12-3 cell growth : Requirement for medium galactosyltransferase activity

Incubation of BALB/3T12-3 cells with uridine diphosphate galactose (UDP-gal) resulted in a concentration-dependent inhibition of cell growth when cells were cultured in calf serum-supplemented Dulbecco's modified Eagle medium (CS-DMEM). Cell growth was completely inhibited by 5 mM UDP-gal with an ID₅₀ of 0.75 mM. This inhibitory effect was reversible. Other nucleotide-sugars, as well as galactose, glucose, and galactose-1-phosphate had no effect on cell growth. UDP-gal had no effect on cell growth when cells were cultured in heat-inactivated calf serum containing DMEM (HICS-DMEM) suggesting that a serum enzyme activity was responsible for the inhibition observed in CS-DMEM. No significant difference could be detected by descending chromatography in the degradation of UDP-gal during 96 h of incubation in CS-DMEM and in HICS-DMEM. Furthermore, the potential breakdown products of UDP-gal had no effect on cell growth when added directly to 3T12 cultures. When cells were incubated with 5 mM UDP-gal+5 mM CDP-choline (a potent inhibitor of pyrophosphatase activity), complete inhibition of cell growth was still observed. However, if cells were incubated with 5 mM UDP-gal+UDP (which inhibited calf serum galactosyltransferase activity), no inhibition of cell growth was observed over that found for UDP alone, suggesting that galactosyltransferase and not pyrophosphatase activity mediated the effect of UDP-gal on cell growth. A direct effect of UDP-gal on cells was suggested by (a) normal growth of cells in UDP-gal-conditioned medium (preincubated with UDP-gal for 24 h followed by dialysis to remove UDP-gal); (b) 3-fold greater incorporation of [³H]galactose from UDP-[³H]gal into cells grown in CS-DMEM than in HICS-DMEM. These studies suggest that the inhibition of 3T12 cell growth by exogenous UDP-gal may be due to alteration of cell surface glycoconjugates by extracellular galactosyltransferase activity.     

Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum     

Mutagenic effect of ultraviolet radiation on the non-acid-fast strain ofMycobacterium phlei

The mutability of the PN strain ofMycobacterium phlei was examined after induction of auxotrophic mutants and of STM and VM-resistant mutants, by UV irradiation. A total of 30 auxotrophic mutants were isolated, most of them amino acid-dependent five purine-dependent, and one uracil-dependent. To induce the mutants higher UV doses had to be used so that the survival of cells in the original suspension would not exceed a few per cent. For further genetic work use can be made of 8 auxotrophic mutants (PN try^?ura^?, PN arg^?ura^?, PN ileu^?val^?, PN ileu^?, PN leu^?, PN lys^?, PN lys^?-VM^r, PN val^?), these showing a low frequency of spontaneous reversions. No spontaneous auxotrophic mutants have been found. The frequency of STM and VM-resistant mutants is increased upon UV irradiation, a post-irradiation incubation in a liquid medium without the drug being essential for their phenotypic expression. The highest increase of the number of these mutants is attained after 48 h of post-irradiation incubation and it has been found that, within a certain experimental scatter, the same frequency increase is found on using a complete or a minimal liquid medium. The frequency of spontaneous STM-resistant mutants lies within 5.8×10^?6–8.8×10^?6, of those VM-resistant between 3.1×10^?5 and 4.1×10^?5. The highest frequency of induced STM-resistant mutants lies between 3.0×10^?5 and 9.3×10^?5 and of VM-resistant mutants between 1.1×10^?4 and 2.2×10^?4     

Novel Temperature-Sensitive Mutants of Escherichia coli That Are Unable To Grow in the Absence of Wild-Type tRNA6Leu

Escherichia coli has only a single copy of a gene for tRNA₆^Leu (Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O²-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA₄^Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O²-methyluridine) as its anticodon, tRNA₆^Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA₆^Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su⁺6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA₆^Leu. These tRNA₆^Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA₄^Leu restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA₄^Leu poorly recognizes the UUG codon at 42°C and that the wild-type tRNA₆^Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA₆^Leu remains to be investigated.In the universal genetic code, 61 sense codons correspond to 20 amino acids, and the various tRNA species mediate the flow of information from the genetic code to amino acid sequences. Since codon-anticodon interactions permit wobble pairing at the third position, 32 tRNAs, including tRNA^fMet, should theoretically be sufficient for a complete translation system. Although some organisms have fewer tRNAs (1), most have abundant tRNA species and multiple copies of major tRNAs. For example, Escherichia coli has 86 genes for tRNA (79 genes identified in reference 14, 6 new ones reported in reference 3, and one fMet tRNA at positions 2945406 to 2945482) that encode 46 different amino acid acceptor species. Although abundant genes for tRNAs are probably required for efficient translation, the significance of the apparently nonessential tRNAs has not been examined.E. coli has five isoaccepting species of tRNA^Leu. According to the wobble rule, tRNA₁^Leu recognizes only the CUG codon. The CUG codon is also recognized by tRNA₃^Leu (tRNA₂^Leu) and thus tRNA₁^Leu may not be essential for protein synthesis. Similarly, tRNA₆^Leu is supposed to recognize only the UUG codon, but tRNA₄^Leu can recognize both UUA and UUG codons. Thus, tRNA₆^Leu appears to be dispensable. The existence of an amber suppressor mutation of tRNA₆^Leu (supP, Su⁺6) supports this possibility. tRNA₆^Leu is encoded by a single-copy gene, leuX (supP), and Su⁺6 has a mutation in the anticodon, which suggests loss of the ability to recognize UUG (26). Why are so many species of tRNA^Leu required? Holmes et al. (12) examined the utilization of the isoaccepting species of tRNA^Leu in protein synthesis and showed that utilization differs depending on the growth medium; in minimal medium, isoacceptors tRNA₂^Leu (cited as tRNA₃^Leu; see Materials and Methods) and tRNA₄^Leu are the predominant species that are found bound to ribosomes, but an increased relative level of tRNA₁^Leu is found bound to ribosomes in rich medium. The existence of tRNA₆^Leu is puzzling. This isoaccepting tRNA accounts for approximately 10% of the tRNA^Leu in total-cell extracts. However, little if any tRNA₆^Leu is found on ribosomes in vivo, and it is also only weakly active in protein synthesis in vitro with mRNA from E. coli (12). It thus appears that tRNA₆^Leu is only minimally involved in protein synthesis in E. coli.To investigate the role of tRNA₆^Leu in E. coli, we attempted to isolate tRNA₆^Leu-requiring mutants from an Su⁺6 strain. These mutants required wild-type tRNA₆^Leu for survival at a nonpermissive temperature. We report here the isolation and the characterization of these mutants.     

The serum growth and survival requirements of SV40-transformed 3T3 cells

Summary Simian virus 40-transformed 3T3 cells are dependent on serum for survival and growth. This growth activity can be separated on a pH 2 Sephadex G100 column into two fractions: a high molecular weight activity and a low molecular weight substance that has recently been characterized as containing as its major agent, biotin. To replace the remainder of the serum requirement, hormones and other growth factors were tested. Both insulin at high, nonphysiological concentrations (200 to 500 ng/ml) and transferrin (5×10⁻⁸ M) stimulate the growth rate in low serum medium (0.3% v/v bovine calf serum DME) individually and, when added together, are nearly as growth enhancing as 10% serum. The need for the residual serum in this medium can be eliminated by the use of crystalline trypsin during trypsinization. Under these serum-free conditions, biotin and transferrin supplementation provide for moderately good growth (20 to 30 hr population doubling time, 1×10⁶ cells/3.2-cm dish final cell density). Insulin addition further stimulates the growth rate (16 to 20 hr) and the final density (1.5×10⁶ cells). Although the protein growth factors, EGF (0.5 to 1.0 ng/ml) and FGF (4 to 10 ng/ml), also appear to enhance growth individually and additively, their effects are slight and very variable. Nevertheless, the complete serum-free medium (DME supplemented with biotin, transferrin, insulin, EGF and FGF) yields growth comparable but still inferior to 10% serum supplementation (14-versus 12-hr population doubling time, 1 to 2×10⁶ versus 2 to 3×10⁶ cells final cell density). This work was supported by NIH Grant CA 20040.     

tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae

tRNAs are highly modified, each with a unique set of modifications. Several reports suggest that tRNAs are hypomodified or, in some cases, hypermodified under different growth conditions and in certain cancers. We previously demonstrated that yeast strains depleted of tRNA^His guanylyltransferase accumulate uncharged tRNA^His lacking the G₋₁ residue and subsequently accumulate additional 5-methylcytidine (m⁵C) at residues C₄₈ and C₅₀ of tRNA^His, due to the activity of the m⁵C-methyltransferase Trm4. We show here that the increase in tRNA^His m⁵C levels does not require loss of Thg1, loss of G₋₁ of tRNA^His, or cell death but is associated with growth arrest following different stress conditions. We find substantially increased tRNA^His m⁵C levels after temperature-sensitive strains are grown at nonpermissive temperature, and after wild-type strains are grown to stationary phase, starved for required amino acids, or treated with rapamycin. We observe more modest accumulations of m⁵C in tRNA^His after starvation for glucose and after starvation for uracil. In virtually all cases examined, the additional m⁵C on tRNA^His occurs while cells are fully viable, and the increase is neither due to the GCN4 pathway, nor to increased Trm4 levels. Moreover, the increased m⁵C appears specific to tRNA^His, as tRNA^Val(AAC) and tRNA^Gly(GCC) have much reduced additional m⁵C during these growth arrest conditions, although they also have C₄₈ and C₅₀ and are capable of having increased m⁵C levels. Thus, tRNA^His m⁵C levels are unusually responsive to yeast growth conditions, although the significance of this additional m⁵C remains unclear.     

Conformational Change of the L-Shaped tRNAPhe Molecule

Abstract

Fluorophore of proflavine was introduced onto the 3′-terminal ribose moiety of yeast tRNA^Phe. The distance between the fluorophore and the fluorescent Y base in the anticodon of yeast tRNA^Phe was measured by a singlet-singlet energy transfer. Conformational changes of tRNA^Phe with binding of tRNA^Glu ₂, which has the anticodon UUC complementary to the anticodon GAA of tRNA^Phe, were investigated. The distance obtained at the ionic strength of 100 mM K⁺ and 10 mM Mg²⁺ is very close to the distance from x-ray diffraction, while the distance obtained in the presence of tRNA^Glu ₂ is significantly smaller. Further, using a fluorescent probe of 4-bromomethl-7-methoxycoumarin introduced onto pseudouridine residue Ψ₅₅ in the TΨC loop of tRNA^Phe, Stern-Volmer quenching experiments for the probe with or without added tRNA^Glu ₂were carried out. The results showed greater access of the probe to the quencher with added tRNA^Glu ₂. These results suggest that both arms of the L-shaped tRNA structure tend to bend inside with binding of tRNA^Glu ₂ and some structural collapse occurs at the corner of the L-shaped structure.     

Structural relationship between tRNA2 Lys and tRNA4 Lys from mouse lymphoma cells

Summary Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNA₂ ^Lys and tRNA₄ ^Lys, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNA₄ ^Lys has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t⁶A^*, replacing t⁶A. This unidentified nucleotide is not a hypomodified form of t⁶A. Thus, tRNA4ys is not a simple precursor of tRNA₂ ^Lys. Both tRNAs have an unidentified nucleotide, U^**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNA₂ ^Lys and tRNA₄ ^Lys were obtained. The distinctions between tRNA₂ ^Lys and tRNA₄ ^Lys may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5,5-P₁,P₄-tetraphosphate, a putative signal in DNA replication.     

Induced changes in the rates of uridine- 3 H uptake and incorporation during the G 1 and S periods of synchronized Chinese hamster cells

The rates of uridine-5-³H incorporation into RNA and the rates of uridine uptake into the acid-soluble pool during the cell cycle of V79 Chinese hamster cells were examined. Cells cultured on Eagle''s minimal essential medium supplemented with fetal calf serum, lactalbumin hydrolysate, glutamine, and trypsin displayed rates of incorporation and uptake which increased only slightly during G₁ and accelerated sharply as DNA synthesis commenced. In contrast, cells cultured on minimal essential medium supplemented only with calf serum exhibited rates of incorporation and uptake which increased linearly through both G₁ and S. The transition from one pattern to the other can be induced within 24 hr and is completely reversible. The nonlinear pattern exhibited by cells grown on the supplemented fetal calf serum medium can also be overcome with high exogenous uridine concentrations. In the presence of 200 µM uridine, these cells display a linear pattern of increase in rates of uridine incorporation and uptake. It is concluded that at lower uridine concentrations the pattern of increase in the rate of uridine incorporation into RNA during the cell cycle for a given population of cells is dependent upon the rate of uridine entry into the cell, and that this pattern is not rigidly determined but can be modified by culture conditions.     

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.     

Combination of the Loss of cmnmU34 with the Lack of sU34 Modifications of tRNA, tRNA, and tRNA Altered Mitochondrial Biogenesis and Respiration

Yeast Saccharomyces cerevisiae MTO2, MTO1, and MSS1 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of carboxymethylaminomethyl (cmnm)⁵s²U₃₄ in mitochondrial tRNA^Lys, tRNA^Glu, and tRNA^Gln. In fact, Mto1p and Mss1p are involved in the biosynthesis of the cmnm⁵ group (cmnm⁵U₃₄), while Mto2p is responsible for the 2-thiouridylation (s²U₃₄) of these tRNAs. Previous studies showed that partial modifications at U₃₄ in mitochondrial tRNA enabled mto1, mto2, and mss1 strains to respire. In this report, we investigated the functional interaction between MTO2, MTO1, and MSS1 genes by using the mto2, mto1, and mss1 single, double, and triple mutants. Strikingly, the deletion of MTO2 was synthetically lethal with a mutation of MSS1 or deletion of MTO1 on medium containing glycerol but not on medium containing glucose. Interestingly, there were no detectable levels of nine tRNAs including tRNA^Lys, tRNA^Glu, and tRNA^Gln in mto2/mss1, mto2/mto1, and mto2/mto1/mss1 strains. Furthermore, mto2/mss1, mto2/mto1, and mto2/mto1/mss1 mutants exhibited extremely low levels of COX1 and CYTB mRNA and 15S and 21S rRNA as well as the complete loss of mitochondrial protein synthesis. The synthetic enhancement combinations likely resulted from the completely abolished modification at U₃₄ of tRNA^Lys, tRNA^Glu, and tRNA^Gln, caused by the combination of eliminating the 2-thiouridylation by the mto2 mutation with the absence of the cmnm⁵U₃₄ by the mto1 or mss1 mutation. The complete loss of modifications at U₃₄ of tRNAs altered mitochondrial RNA metabolisms, causing a degradation of mitochondrial tRNA, mRNA, and rRNAs. As a result, failures in mitochondrial RNA metabolisms were responsible for the complete loss of mitochondrial translation. Consequently, defects in mitochondrial protein synthesis caused the instability of their mitochondrial genomes, thus producing the respiratory-deficient phenotypes. Therefore, our findings demonstrated a critical role of modifications at U₃₄ of tRNA^Lys, tRNA^Glu, and tRNA^Gln in maintenance of mitochondrial genome, mitochondrial RNA stability, translation, and respiratory function.     

Cocrystallization of Lysyl–tRNA synthetase from Thermus thermophilus with its cognate tRNAlys and with Escherichia coli tRNAlys

Lysyl-tRNA synthetase from Thermus thermophilus has been cocrystallized with either its cognate tRNA^lYS or Escherichia coli tRNA^lys using ammonium sulfate as precipitant. The crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA in 18–22% ammonium sulfate in 50 mM Tris-maleate buffer at pH 7.5. Both complexes form square prismatic, tetragonal crystals with very similar unit cell parameters (a = b = 233 Å, c = 119 Å) and diffract to at least 2.7 Å resolution. However the homocomplex is of space group P42₁2 and the heterocomplex of space group I422. © 1995 Wiley-Liss, Inc.