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1.
The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-cellulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNACys, tRNATrp, tRNATyr, a major peak of tRNAPhe, and a large fraction of tRNALeu were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNASer species and with minor tRNALeu species. In addition, several anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected.  相似文献   

2.
Lambda bacteriophage containing yeast tyrosine transfer RNA genes were prepared by molecular recombination. These phage were identified by hybridization of 125I-labeled yeast tRNATyr to plaques from lambda-yeast recombinant phage pools. The cloned yeast EcoRI fragments that hybridize to 125I-labeled tRNATyr were compared in size with the fragments in total yeast DNA that hybridize to the same probe. These comparisons indicate that seven of the eight different tRNATyr genes have been isolated. Unambiguous evidence that these seven fragments contain tRNATyr coding regions was obtained by showing that they hybridize to aminoacylated [3H]Tyr-tRNATyr. Only one of the fragments hybridizes to 32P-labeled total yeast tRNA in the presence of competing unlabeled tRNATyr; the tRNATyr genes, therefore, are not predominantly organized into heteroclusters of tRNA genes.  相似文献   

3.
Sensory ataxic neuropathy (SAN) is a recently identified neurological disorder in golden retrievers. Pedigree analysis revealed that all affected dogs belong to one maternal lineage, and a statistical analysis showed that the disorder has a mitochondrial origin. A one base pair deletion in the mitochondrial tRNATyr gene was identified at position 5304 in affected dogs after re-sequencing the complete mitochondrial genome of seven individuals. The deletion was not found among dogs representing 18 different breeds or in six wolves, ruling out this as a common polymorphism. The mutation could be traced back to a common ancestor of all affected dogs that lived in the 1970s. We used a quantitative oligonucleotide ligation assay to establish the degree of heteroplasmy in blood and tissue samples from affected dogs and controls. Affected dogs and their first to fourth degree relatives had 0–11% wild-type (wt) sequence, while more distant relatives ranged between 5% and 60% wt sequence and all unrelated golden retrievers had 100% wt sequence. Northern blot analysis showed that tRNATyr had a 10-fold lower steady-state level in affected dogs compared with controls. Four out of five affected dogs showed decreases in mitochondrial ATP production rates and respiratory chain enzyme activities together with morphological alterations in muscle tissue, resembling the changes reported in human mitochondrial pathology. Altogether, these results provide conclusive evidence that the deletion in the mitochondrial tRNATyr gene is the causative mutation for SAN.  相似文献   

4.
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 tRNATyr. A second chromatography system for separating these forms of tRNATyr 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 tRNATyr in a ratio of 4060, the suppressor mutant in a ratio of 6040. 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 tRNATyr 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 tRNATyr. Storage results in the loss of the enzyme's ability to discriminate against the second form of tRNATyr 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 tRNATyr 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.  相似文献   

5.
Further investigations into the properties of the mercury derivative formed by the reaction of 4-thiouridine-containing tRNAs and pentafluorophenylmercury chloride have been carried out. tRNAfMet (which contains only one 4-thiouridine residue) has been isolated by a one-step column Chromatographic procedure from unfractionated Escherichia coli tRNA and has been shown to react with the mercury compound to give a derivative which has similar properties to those previously reported for the corresponding mercury derivative of tRNATyr which contains two adjacent 4-thiouridine residues. The mercury derivative of tRNATyr appears to be a competitive inhibitor of tRNATyr in the aminoacylation reaction (tRNATyrKm = 0.42 μM, mercury derivative of tRNATyrKi = 0.11 μM). The mercury derivative of Tyr-tRNATyr can be made, but only by the reaction of the mercury compound with the aminoacylated tRNA.  相似文献   

6.
Escherichia coli DNA and fragmented rRNA were used as a model system to study the effect of RNA fragment size in hybridization-competition experiments. Though no difference in hybridization rates was observed, the relative stabilities of the RNA/DNA hybrids were found to be largely affected by the fragment size of the RNA molecule. Intact rRNA was shown to replace shorter homologous rRNA sequences in their hybrids, the rate of the displacement being dependent on the molecular size of the RNA fragments. Hybridization-competition experiments between molecules of different lengths are expected to be complicated by the displacement reaction. The synthesis of tRNATyr-like sequences transcribed in vitro on φ80psu3+ bacteriophage DNA was measured by hybridization competition assays. Indirect competition with labelled E. coli tRNATyr hybridization revealed that the in vitro-synthesized RNA contained significant amounts of tRNATyr; these sequences could not, however, be detected by the direct competition method in which labelled in vitro-synthesized RNA competes with E. coli tRNATyr for hybridization to φ80psu3+ DNA. These contradictory results can be traced to the differences in size of the competing molecules in the hybridization-competition reaction. Indeed, in vitro-transcribed tRNATyr-like sequences, longer than mature tRNA, were found to displace efficiently E. coli tRNATyr from its hybrids with φ80psu3+ DNA. These findings explain why such sequences could not be detected by direct competition with E. coli tRNATyr.  相似文献   

7.
The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNASer(CGA), an Arabidopsis intron-containing tRNATyr(GTA) and an Arabidopsis intron-containing tRNAMet(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of β-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNASer gene and the GUS reporter gene resulted in ~10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNATyr or tRNAMet genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNAMet(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNATyr to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNATyr and tRNAMet were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.  相似文献   

8.
The anticodon-anticodon complex   总被引:6,自引:0,他引:6  
Gel electrophoresis has been used to measure the binding between two tRNAs with complementary anticodons, tRNAVal (Escherichia coli) (anticodon X,A,C) and tRNATyr (E. coli) (anticodon Q,U,A). The association constant K at 0 °C was found to be 4 × 105 m?1 which is about three orders of magnitude greater than the association constant for tRNATyr (E. coli) binding its trinucleotide codon UAC. The temperature dependence of K suggests that this results from the rigidity of the anticodon loop. tRNATyr (E. coli) binds an order of magnitude more weakly to tRNAVal (yeast) than to tRNAVal (E. coli), presumably because it contains the wobble base pair A · I. The relationship between the anticodon-anticodon complex and codon recognition is discussed.  相似文献   

9.
The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNATyrs. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNATyr(GΨA). Structural information for TyrRS–tRNATyr complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs–tRNATyrs pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNATyr recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNATyr pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNATyr. On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.  相似文献   

10.
11.
Translation of tobacco mosaic virus (TMV) RNA in tobacco protoplasts yields the 17.5-K coat protein, a 126-K protein and a 183-K protein which is generated by an efficient readthrough over the UAG termination codon at the end of the 126-K cistron. In wheat germ extracts, however, only the 5'-proximal 126-K cistron is translated whereas the 183-K readthrough protein is not synthesized. Purification and sequence analysis of the endogenous tyrosine tRNAs revealed that the uninfected tobacco plant contains two tRNAsTyr, both with GΨA anticodons which stimulate the UAG readthrough in vitro and presumably in vivo. In contrast, ˜85% of the tRNATyr from wheat germ contains a QΨA anticodon and ˜15% has a GΨA anticodon. Otherwise the sequences of tRNAsTyr from wheat germ and tobacco are identical. UAG readthrough and hence synthesis of the 183-K protein is only stimulated by tRNATyrGΨA and not at all by tRNATyrQΨA. The tRNAsTyr from wheat leaves were also sequenced. This revealed that adult wheat contains tRNATyrGΨA only. This is very much in contrast to the situation in animals, where Q-containing tRNAs are characteristic for adult tissues whereas Q deficiency is typical for the neoplastic and embryonic state.  相似文献   

12.
Dietary cadmium causes the queuine-containing, Q(+), isoacceptors to increase relative to the guanine-containing, Q(?), ones of tRNATyr, tRNAHis and tRNAAsp of Drosophila melanogaster. Of the other divalent cations examined, Sr2+, Ni2+, Cu2+, Zn2+ and Hg2+, only Hg2+ failed to cause an increase in Q(+)tRNATyr. For these results, all pre-adult stages of the organism were spent on media containing the divalent ions. Adult flies that had developed on a normal diet also responsed to divalent ions; Hg2+ as well as Cd2+, Sr2+ and Zn2+ caused an increase in Q(+)tRNATyr in 4 days. Using adult flies, the rate of the response was measured; when placed on a Cd2+-containing diet, they formed significantly more Q(+)tRNATyr within 24 h as compared to adults on a normal diet. Whether the queuine is derived from the diet or from de novo synthesis is yet to be determined. Since the metal ions represent a range of values in the ‘hard-soft’ classification, different sites of reaction are expected, yet for Drosophila a common result is an alteration in the ratio of Q(+) and Q(?) isoacceptors of these tRNAs. The transition to Q(+)tRNA may be an early indication of the metabolic imbalances resulting from the presence of the divalent cation.  相似文献   

13.
Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNATyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNATyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNATyr. The endogenous TyrRS and tRNATyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNATyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.  相似文献   

14.
Total tRNA of Chlamydomonas reinhardii was fractionated by 2-dimensional gel electrophoresis. Sixteen tRNAs specific for eleven amino acids could be identified by aminoacylation with Escherichia coli tRNA synthetases. Hybridization of these tRNAs with chloroplast restriction fragments allowed for the localization of the genes of tRNATyr, tRNAPro, tRNAPhe (2 genes), tRNAIle (2 genes) and tRNAHis (2 genes) on the chloroplast genome of C. reinhardii. The genes for tRNAAla (2 genes), tRNAAsn and tRNALeu were mapped by using individual chloroplast tRNAs from higher plants as probes.  相似文献   

15.
Summary The nucleotide sequences of the mitochondrial origin of light-strand replication and the five tRNA genes surrounding it were determined for three marsupials. The region was found to be rearranged, leaving only the tRNATyr gene at the same position as in placental mammals andXenopus. Distribution of the same rearranged genotype among two marsupial families indicates that the events causing the rearrangements took place in an early marsupial ancestor. The putative mitochondrial light-strand origin of replication in marsupials contains a hairpin structure similar to other vertebrate origins and, in addition, extensive flanking sequences that are not found in other vertebrates. Sequence comparisons among the marsupials as well as placentals indicate that the tRNATyr gene has been evolving under more constraints than the other tRNA genes.Deceased July 21, 1991  相似文献   

16.
Mitochondrial glutamyl-tRNA isolated from mitochondria of Saccharomyces cerevisiae was separated into two distinct species by re versed-phase chromatography. The migration of the two mitochondrial glutamyl-tRNAs (tRNAIGlu and tRNAIIGlu) differed from that of two glutamyl-tRNA species found in the cytoplasm of a mitochondrial DNA-less petite strain. Both mitochondrial tRNAs hybridized with mitochondrial DNA. Three lines of evidence demonstrate that mitochondrial tRNAIGlu and tRNAIIGlu are transcribed from different mitochondrial cistrons. First the level of hybridization of a mixture of the two tRNAs to mitochondrial DNA was equal to the sum of the saturation hybridization levels of each glutamyl-tRNA alone. Second, the two mitochondrial glutamyl-tRNAs did not compete with each other in hybridization competition experiments. Finally the tRNAs showed individual hybridization patterns with different petite mitochondrial DNAs.Hybridization of the tRNAs to mitochondrial DNA of genetically defined petite strains localized each tRNA with respect to antibiotic resistance markers. The two glutamyl-tRNA cistrons were spatially separated on the genetic map.  相似文献   

17.
Primary structure of Bacillus subtilis tRNAsTyr   总被引:4,自引:0,他引:4  
tRNAITyr and tRNAIITyr have been purified from B.subtilis and their nucleotide sequence determined. tRNAITyr differs from tRNAIITyr only by the extent of modification of the adenosine in 3′ position adjacent to the anticodon, i6A and ms2i6A respectively.  相似文献   

18.
Tobacco tRNATyr genes are mainly organized as a dispersed multigene family as shown by hybridization with a tRNATyr-specific probe to Southern blots of Eco RI-digested DNA. A Nicotiana genomic library was prepared by Eco RI digestion of nuclear DNA, ligation of the fragments into the vector gtWES·B and in vitro packaging. The phage library was screened with a 5-labelled synthetic oligonucleotide complementary to nucleotides 18 to 37 of cytoplasmic tobacco tRNATyr. Eleven hybridizing Eco RI fragments ranging in size from 1.7 to 7.5 kb were isolated from recombinant lambda phage and subcloned into pUC19 plasmid. Four of the sequenced tRNATyr genes code for the known tobacco tRNA1 Tyr (GA) and seven code for tRNA2 Tyr (GA). The two tRNA species differ in one nucleotide pair at the basis of the TC stem. Only one tRNATyr gene (pNtY5) contains a point mutation (T54A54). Comparison of the intervening sequences reveals that they differ considerably in length and sequence. Maturation of intron-containing pre-tRNAs was studied in HeLa and wheat germ extracts. All pre-tRNAsTyr-with one exception-are processed and spliced in both extracts. The tRNATyr gene encoded by pNtY5 is transcribed efficiently in HeLa extract but processing of the pre-tRNA is impaired.  相似文献   

19.
Transfer RNATyr (anticodon GA) was isolated from Drosophila melanogaster by means of Sepharose 4B, RPC-5, and polyacrylamide gel electrophoresis. The tRNA was iodinated in vitro with Na125I and hybridized in situ to salivary gland chromosomes from Drosophila. The genes of tRNATyr were localized in eight regions of the genome by autoradiography. Restriction enzyme analysis of genomic DNA indicated that the haploid Drosophila genome codes for about 23 tRNATyr genes. The regions 22F and 85A each contain four to five tRNATyr genes, whereas the regions 28C, 41AB, 42A, 42E, and 56D each contain two to three tRNATyr genes.  相似文献   

20.
Import of nucleus-encoded tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania involves recognition of specific import signals by the membrane-bound import machinery. Multiple signals on different tRNA domains may be present, and further, importable RNAs interact positively (Type I) or negatively (Type II) with one another at the inner membrane in vitro. By co-transfection assays, it is shown here that tRNATyr (Type I) transiently stimulates the rate of entry of tRNAIle (Type II) into Leishmania mitochondria in transfected cells, and conversely, is inhibited by tRNAIle. Truncation and mutagenesis experiments led to the co-localization of the effector and import activities of tRNATyr to the D domain, and those of tRNAIle to the variable region–T domain (V-T region), indicating that both activities originate from a single RNA–receptor interaction. A third tRNA, human tRNALys, is imported into Leishmania mitochondria in vitro as well as in vivo. This tRNA has Type I and Type II motifs in the D domain and the V-T region, respectively, and shows both Type I and Type II effector activities. Such dual-type tRNAs may interact simultaneously with the Type I and Type II binding sites of the inner membrane import machinery.  相似文献   

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