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1.
1H NMR of valine tRNA modified bases. Evidence for multiple conformations. 总被引:1,自引:1,他引:0
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Methyl and methylene protons of dihydrouridine 17 (hU), 6-methyladenosine 37 (M6A), 7-methylguanosine 46 (m7G), and ribothymidine 54 (rT) give clearly resolved peaks (220 MHz) for tRNA1val (coli solutions in D2O, 0.25 m NaCl, at 27 degrees C. Chemical shifts are generally consistent with a solution structure of tRNA1val similar to the crystal structure of tRNAphe (yeast). At least 3 separate transitions are observed as the temperature is raised. The earliest involves disruption of native tertiary structure and formation of intermediate structures in the m7G and rT regions. A second transition results in a change in structure of the anticodon loop, containing m6A. The final step involves unfolding of the m7G and rT intermediates and melting of the TpsiC helix. Low salt concentrations produce multiple, partially denatured conformations, rather than a unique form, for tRNA1val. Native structure is almost completely reformed by addition of Na+ but Mg2+ is required for correct conformation in the vicinity of m7G. 相似文献
2.
Binding and cleavage of pre-tRNA by the Xenopus splicing endonuclease: two separable steps of the intron excision reaction 总被引:4,自引:0,他引:4
We have constructed three base-substitution mutants of the yeast tRNALeu3 gene. In two of them the ability to form an extended anticodon stem is lost. In the first mutant the bases encoding the anticodon change from TTG to GAC (positions 37, 36, 35); in the second, the nucleotides encoding the region of the intron that base-pair with the anticodon change from CAA to GTC (positions 48, 47, 46). The third is a double mutant characterized by both substitutions described above so that its ability to form an extended anticodon stem is restored. The precursors derived from the two single mutants are accurately spliced in the X. laevis germinal vesicles (GV) extract: pairing of the anticodon with the intron, therefore, is not required for the splicing reaction. The precursor derived from the double mutant is not spliced, indicating that the new extended anticodon stem exerts an inhibitory action. Since the double mutant precursor binds to the purified splicing endonuclease, binding and cleavage occur as two separable steps in the intron excision reaction. 相似文献
3.
Escherichia coli supH suppressor: temperature-sensitive missense suppression caused by an anticodon change in tRNASer2. 总被引:12,自引:5,他引:7
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S Thorbjarnardttir H Uemura T Dingermann T Rafnar S Thorsteinsdttir D Sll G Eggertsson 《Journal of bacteriology》1985,161(1):207-211
We describe the cloning and the DNA sequence of the Escherichia coli supH missense suppressor and of the supD60(Am) suppressor genes. supH is a mutant form of serU which codes for tRNASer2. The supH coding sequence differs from the wild-type sequence by a single nucleotide change which corresponds to the middle position of the anticodon. The CGA anticodon of wild-type tRNA and CUA anticodon of supD tRNA is changed to CAA in supH tRNA, which is expected to recognize the UUG leucine codon. We propose that the supH suppressor causes the insertion of serine in response to this codon. The temperature sensitivity caused by supH may be due to a conformation of the CAA anticodon in the supH tRNASer that is slightly different than that in the corresponding tRNALeu species. 相似文献
4.
O O Favorova T A Zargarova V S Rukosuyev S F Beresten L L Kisselev 《European journal of biochemistry》1989,184(3):583-588
The content of Trp-tRNA synthetase in pancreas and liver of cattle, sheep, swine, rat, rabbit and man was assayed by direct radioimmunoblotting with a 125I-labelled monoclonal antibody Am1, specifically interacting with any eukaryotic Trp-tRNA synthetase. Its content in the organs studied, with the exception of bovine and sheep pancreas, was found to be 0.002-0.012% of total proteins. The enzyme content in bovine pancreas was about 0.2% of total proteins, i.e. 70 times higher than in bovine liver; similar correlations were found for sheep. The Trp-tRNA synthetase levels in each organ varied from animal to animal of the same species by not more than a factor of four; these individual variations cannot affect the conclusion about the profound differences in the levels of the enzyme in pancreases of Ruminantia and of the other mammalians. As shown by indirect immunofluorescence technique, bovine Trp-tRNA synthetase is mainly located in the exocrine part of the pancreas. Moreover, the immunoreactive material is detectable also in bovine (not human) pancreatic juice. The abnormally high Trp-tRNA synthetase content in the ruminant pancreas may be connected with unknown function(s) of this protein somehow related to the peculiarities of digestion of these mammals. 相似文献
5.
Effect of ribosome binding and translocation on the anticodon of tRNAPhe as studied by wybutine fluorescence. 总被引:2,自引:1,他引:1
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The complexes of N-AcPhe-tRNAPhe (or non-aminoacylated tRNAPhe) from yeast with 70S ribosomes from E. coli have been studied fluorimetrically utilizing wybutine, the fluorophore naturally occurring next to the 3' side of the anticodon, as a probe for conformational changes of the anticodon loop. The fluorescence parameters are very similar for tRNA bound to both ribosomal sites, thus excluding an appreciable conformational change of the anticodon loop upon translocation. The spectral change observed upon binding of tRNAPhe to the P site even in the absence of poly(U) is similar to the one brought about by binding of poly(U) alone to the tRNA. This effect may be due to a hydrophobic binding site of the anticodon loop or to a conformational change of the loop induced by binding interactions of various tRNA sites including the anticodon. 相似文献
6.
The influence of elongation-factor-Tu . GTP and anticodon-anticodon interactions on the anticodon loop conformation of yeast tRNATyr 总被引:2,自引:0,他引:2
The interactions of yeast tRNATyr, spin-labelled at position i6A-37 next to the anticodon, with EF-Tu . GTP and with Escherichia coli tRNAVal (which has a complementary anticodon) have been studied. The immobilization of the spin label upon ternary complex formation shows a conformational change of the anticodon region, although this part of tRNATyr is not in direct contact with the protein, as indicated by RNase T1 digestion. Upon anticodon-anticodon interaction, no conformational change of the anticodon loop of tRNATyr was observed. 相似文献
7.
F V Tuzikov N A Tuzikova V I Vavilin V V Zinov'ev E G Malygin O O Favorova T A Zargarova M A Sudomoina L L Kiselev 《Molekuliarnaia biologiia》1991,25(3):740-751
By means of small angle X-ray scattering, an aggregation of beef pancreas Trp-tRNA synthetase (EC 6.1.1.2) was observed at physiological temperatures. A Trp-tRNA synthetase preparation which is homogeneous after PAGE in beta-ME-SDS was found to be heterogeneous in particle sizes even at low (4-8 degrees C) temperature. At heating up to 30-45 degrees C, the oligomer sizes increased as well as its proportion depending on the incubation time and temperature; very large aggregates were observed 10 times exceeding the sizes of initial particles. Cooling to 20 degrees C caused no disaggregation due to disulphide bond formation between associated subunits of Trp-tRNA synthetase. A hypothesis is proposed that the aggregation of bovine Trp-tRNA synthetase evaluated in vitro and not observed earlier with any aminoacyl-tRNA synthetases of unicellular organisms might serve as one of the mechanisms of its compartmentation in pancreas. 相似文献
8.
Oligonucleotide binding to the native and denatured conformers of yeast transfer RNA-3 Lea 总被引:2,自引:0,他引:2
The equilibrium binding patterns of complementary oligonucleotides to the native and denatured conformers of yeast transfer RNA3Leu have been determined. The pattern of binding to the native conformer follows that observed previously with other tRNAs. The results indicate that the anticodon loop and 3′ terminus are free in solution, and that all stems of the cloverleaf appear intact, although the dihydrouracil and “extra arm” stems are sufficiently weak to be subject to competitive binding by the probe oligomers. The T ΨC loop is also inaccessible to oligomer binding, while the dihydrouracil loop shows a low level of binding suggestive of oligomer competition with existing RNA structure. By contrast, in the denatured conformer the dihydrouracil loop and stem show strong oligomer binding characteristics of random RNA segments, whereas the anticodon loop no longer binds complementary oligomers. Binding to other regions remains unchanged, suggesting that the three major cloverleaf stems are intact. These observations are used as a basis for consideration of models for the two conformers. 相似文献
9.
Initiator tRNAs have an anticodon loop conformation distinct from that of elongation tRNAs as detected by susceptibility to S1 nuclease. We now find the anticodon loop conformation of E. coli tRNAfMet to be stable under different salt conditions as detected by using S1 nuclease as a structural probe. In contrast, a conformational change is observed in the T- and D- loop of this tRNA in the absence of added Mg2+. This change can be suppressed by spermine. Even under those conditions effecting a change in T- and D- loop conformation, the anticodon loop does not change. This suggests that the conformational shift is controlled by Mg2+ and restricted to the D- and T- loop region only without affecting the anticodon domain. The use of S1 nuclease as a conformational probe requires the use of kinetic studies to determine the initial cleavage sites. Thus, the use of a strong inhibitor which immediately stops the action of this nuclease is necessary. ATP is shown to be such an inhibitor. 相似文献
10.
Aminoacyl-tRNA (AA-tRNA) formation is a key step in protein biosynthesis. This reaction is catalyzed with remarkable accuracy by the AA-tRNA synthetases, a family of 20 evolutionarily conserved enzymes. The lack of cysteinyl-tRNA (Cys-tRNA) synthetase in some archaea gave rise to the discovery of the archaeal prolyl-tRNA (Pro-tRNA) synthetase, an enzyme capable of synthesizing Pro-tRNA and Cys-tRNA. Here we review our current knowledge of this fascinating process. 相似文献
11.
A single peak of tRNAGlu is obtained upon chromatography of unfractionated tRNA from Escherichia coli on DEAE-Sephadex A-50 if this tRNA was previously renatured, whereas two peaks of tRNAGlu are resolved if the sample chromatographed is a mixture of native (renatured) and denatured tRNA. Higher resolution analysis of native E. coli tRNA by RPC-5 chromatography showed that most of the tRNAGlu is present in one peak, eluted shortly after a minor peak containing about or less than 5% of the total amount of tRNAGlu; these two peaks were also observed with commercially available tRNAGlu purified from E. coli. When denatured, the tRNAGlu present in each of these two peaks was eluted from the RPC-5 column at a much lower salt concentration. The properties of the denatured conformers obtained from native tRNAGlu present in the major and minor peaks, and the variation, with growth conditions of E. coli, in the relative amount of tRNAGlu in the minor peak suggest that the tRNAGlu present in the minor peak is an undermodified form of the tRNAGlu present in the major peak. This tRNAGluUUC (or tRNAGluSUC when modified in the anticodon) would then be the only tRNA species acceptor of glutamate in E. coli. 相似文献
12.
Electron microscopy of simian virus 40 DNA configuration under denaturation conditions. 总被引:2,自引:2,他引:0
After isolation, the DNA of simian virus 40 appeared as a negative supertwist (form I) or as an open circle with at least one single-strand scission (form II). Under the denaturation conditions usually applied, such as heating in the presence of formaldehyde or application of alkali, form I molecules could appear as "relaxed" circles without single-strand scissions (form I') containing denatured sites not visible under the electron microscope. Form II molecules, under these denaturation conditions, showed partial or complete strand separations allowing the construction of denaturation maps. By using a modified denaturation procedure, i.e., heating of isolated SV40 DNA in the presence of dimethyl sulfoxide and formaldehyde followed by keeping the DNA in this denaturation solution at room temperature for periods up to 3 weeks, partially denatured relaxed circles without single-strand scissions were produced (form I'D) in addition to completely denatured form II molecules. The absence of single-strand scissions in form I'D molecules was demonstrated by a second heat treatment, which did not change the configuration of this molecular form. Form I'D molecules, in contrast to form I', contained denatured sites clearly discerible under the electron microscope. This combined application of two subsequent denaturation steps (denaturation by heating followed by denaturation at room temperature and neutral pH) showed that the molecular configuration I'D originated in two steps. The heating procedure produced molecules not distinquishable by electron microscopy from form I. In contrast to form I, these molecules were assumed to possess "preformed" denaturation sites (form I). Further treatment of form I molecules with denaturation solution at room temperature finally transformed them into convalently closed, relaxed, partially denatured circles exhibiting strand separations easily measurable on electron micrographs (form I'D). Denaturation maps of form I'D molecules were constructed by computer and compared with denaturation maps derived from partially denatured form II molecules. From these denaturation maps it can be concluded that the melting of base pairs occurring during the transition of simian virus 40 DNA form I into form I'D also preferentially happened at sites rich in the bases adenosine and thymine. 相似文献
13.
Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli. 总被引:12,自引:6,他引:6
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We introduced into a stringent Escherichia coli tryptophan auxotroph a plasmid bearing the tRNA(Trp) gene under the control of an inducible promoter. This allows us to manipulate the total concentration of tRNA(Trp) in the cell according to whether and when inducer is added to the culture. We also manipulated the concentration of Trp-tRNA(Trp) in vivo since the strain used bears a mutation in the Trp-tRNA synthetase affecting the Km for tryptophan, such that varying the exogenous concentration of tryptophan led to variation in the level of Trp-tRNA(Trp) in the cell. With this system, we found that the signal eliciting ppGpp synthesis during a stringent response triggered by tryptophan limitation did not depend on the absolute concentration of either charged or uncharged tRNA(Trp) but rather depended on a decline in the ratio of charged/uncharged tRNA(Trp). In addition, we found that the amplitude of the response, once triggered by tryptophan limitation, was determined by the total concentration of tRNA(Trp) present in the cell (which is mostly uncharged at that point in time). However, excess uncharged tRNA(Trp) did not amplify ppGpp synthesis triggered by limitation of a different amino acid. These data provide in vivo support for the in vitro-derived model of ppGpp synthesis on ribosomes. 相似文献
14.
The authors in a previous report (Klausner, R. D., Kempf, C., Weinstein, J. N., Blumenthal, R., and van Renswoude, J. (1983) Biochem. J. 212, 801-810) have argued that native folding of ovalbumin occurs during translation, but not in a renaturation system of the denatured form. To re-examine the possibility, we searched for the conditions of correct oxidative refolding of denatured disulfide-reduced ovalbumin. Data of trypsin resistance, CD-spectrum, and selective reactivity of cysteine sulfhydryls revealed that the fully denatured protein can refold into the native conformation under disulfide-reduced conditions. The interconversion between the native and denatured forms was fully reversible with a free energy change for unfolding of 6.6 kcal/mol at 25 degrees C. Subsequent reoxidation under a variety of redox conditions generated only one disulfide bond in the reduced refolded protein with six cysteine sulfhydryls. Furthermore, the regenerated disulfide was found by peptide analyses to correspond to the native disulfide pairing, Cys73-Cys120. We, therefore, concluded that co-translational folding, if any, is not requisite for the correct oxidative folding of ovalbumin. 相似文献
15.
16.
The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease. 总被引:8,自引:0,他引:8
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The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins. 相似文献
17.
The kinetics of binding of U-U-C-A to a dodecanucleotide anticodon fragment from yeast tRNA-Phe. 总被引:1,自引:0,他引:1
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The kinetics of U-U-C-A binding to the dodecanucleotide (A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-Gp) isolated from the anticodon region of yeast tRNA-Phe are similar to the kinetics of binding of U-U-C-A to intact tRNA-Phe. A large enhancement in binding constant over that predicted for U-U-C-A-U-G-A-A is observed for both the complexes of dodecanucleotide and tRNA-Phe with U-U-C-A. This strongly suggests that both the anticodon loop in tRNA-Phe and the dodecanucleotide can form four base pairs with U-U-C-A. Furthermore, the enhanced stability cannot be attributed to a special conformation of the anticodon loop, but instead the anticodon loop is probably flexible. A likely explanation for the increased binding is the effect of non-base-paired ends. This increased thermodynamic stability comes from a larger entropy gain rather than a larger enthalpy decrease. 相似文献
18.
Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E. coli: a potential application for the purification of the enzyme.
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E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp. However, in the presence of Mg++, only tRNATrp can dissociate the complex whereas ATP has no effect. E. coli total tRNA or tRNAMet, at the same concentration, cannot displace the synthetase from the complex. It is suggested that the Blue-dextran binds to the synthetase through its tRNA binding domain. This hypothesis is supported by previous findings with polynucleotide phosphorylase showing that Blue-dextran Sepharose can be used in affinity chromatography to recognize a polynucleotide binding site of the protein. The selective elution by its cognate tRNA of Trp-tRNA synthetase bound to Blue-dextran Sepharose provides a rapid and efficient purification of the enzyme. Examples of other synthetases and nucleotidyl transferases are also discussed. 相似文献
19.
The total nucleotide sequence of tRNAGGA/G -Gly2 from Escherichia coli is pG-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-C-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-AOH, where T- at position 53 is ribothymidylic acid, and psi- at position 54 is pseudouridylic acid; N- at position 36 is an unidentified derivative of uridylic acid, and is present in modified form in a portion of tRNAGGA/G -Gly 2 molecules isolated from E. coli cells. The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNAGGA/G -Gly 2 (nucleotide position 38). A secondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNAsuA36(HA), -Gly 2 suggesting that the enzymes responsible for this modification recognize the anticodon sequences of prospective tRNA substrates. The creation of a missense-suppressing tRNA, tRNAsuA36(HA), -Gly 2 by an alteration of the anticodon sequence of tRNAGGA/G -Gly 2 is analogous to mechanisms whereby other suppressor tRNAs have arisen. The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions of four glycine isoaccepting tRNAs specified by E. coli and bacteriophage T4 suggests that these regions may be recognized by the glycyl-tRNA synthetase; the involvement of the anticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNAsuA36(HA) -Gly 2. 相似文献
20.
Mutant tRNAs containing an extra nucleotide in the anticodon loop are known to suppress +1 frameshift mutations, but in no case has the molecular mechanism been clarified. It has been proposed that the expanded anticodon pairs with a complementary mRNA sequence (the frameshift sequence) in the A site, and this quadruplet "codon-anticodon" helix is translocated to the P site to restore the correct reading frame. Here, we analyze the ability of tRNA analogs containing expanded anticodons to recognize and position mRNA in ribosomal complexes in vitro. In all cases tested, 8 nt anticodon loops position the 3' three-quarters of the frameshift sequence in the P site, indicating that the 5' bases of the expanded anticodon (nucleotides 33.5, 34, and 35) pair with mRNA in the P site. We also provide evidence that four base-pairs can form between the P-site tRNA and mRNA, and the fourth base-pair involves nucleotide 36 of the tRNA and lies toward (or in) the 30 S E site. In the A site, tRNA analogs with the expanded anticodon ACCG are able to recognize either CGG or GGU. These data imply a flexibility of the expanded anticodon in the A site. Recognition of the 5' three-quarters of the frameshift sequence in the A site and subsequent translocation of the expanded anticodon to the P site results in movement of mRNA by four nucleotides, explaining how these tRNAs can change the mRNA register in the ribosome to restore the correct reading frame. 相似文献