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1.
Binding of Mn2+ to the whole molecule, fragments and complementary fragment recombinations of yeast tRNAPhe, 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 tRNAPhe permits reasonable conclusions concerning the nature and location of the various classes of sites on tRNAPhe. Binding of Mn2+ to intact tRNAPhe 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 tRNAPhe fragments (5′-12, 3′-12, 5′-35, 3′-25), 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 tRNAPhe 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.  相似文献   

2.
Abstract

Fluorophore of proflavine was introduced onto the 3′-terminal ribose moiety of yeast tRNAPhe. The distance between the fluorophore and the fluorescent Y base in the anticodon of yeast tRNAPhe was measured by a singlet-singlet energy transfer. Conformational changes of tRNAPhe with binding of tRNAGlu 2, which has the anticodon UUC complementary to the anticodon GAA of tRNAPhe, 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 tRNAGlu 2 is significantly smaller. Further, using a fluorescent probe of 4-bromomethl-7-methoxycoumarin introduced onto pseudouridine residue Ψ55 in the TΨC loop of tRNAPhe, Stern-Volmer quenching experiments for the probe with or without added tRNAGlu 2were carried out. The results showed greater access of the probe to the quencher with added tRNAGlu 2. These results suggest that both arms of the L-shaped tRNA structure tend to bend inside with binding of tRNAGlu 2 and some structural collapse occurs at the corner of the L-shaped structure.  相似文献   

3.
A chloroplast tRNAmMet species from Scenedesmusobliquus is very poorly 5′-end [32P] 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 tRNAGlu.  相似文献   

4.
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 (tRNAiMet); Class B contains tRNA in which one mole of a modified uridine (rT, ψ, or 2′-O-methylribothymidine) is found per mole of tRNA (tRNASer, tRNATrp, and tRNALys, respectively). Class C contains tRNA in which there is a partial conversion of uridine to ribothymidine (tRNAPhe, tRNA1Gly, tRNA2Gly); Class D contains tRNA which totally lacks ribothymidine (tRNAVal). Only those tRNAs in Class C are acceptable substrates for E.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.  相似文献   

5.
Three-dimensional atomic models of complexes between yeast tRNAPhe and 10- or 15-mer oligonucleotides complementary to the 3′-terminal tRNA sequence have been constructed using computer modeling. It has been found that rapidly formed primary complexes appear when an oligonucleotide binds to the coaxial acceptor and T stems of the tRNAPhe along the major groove, which results in the formation of a triplex. Long stems allow the formation of a sufficiently strong complex with the oligonucleotide, which delivers its 3′-terminal nucleotides to the vicinity of the T loop adjoining the stem. These nucleotides destabilize the loop structure and initiate conformational rearrangements involving local tRNAPhe destruction and formation of the final tRNAPhe-oligonucleotide complementary complex. The primary complex formation and the following tRNAPhe destruction constitute the “molecular wedge” mechanism. An effective antisence oligonucleotide should consist of three segments—(1) complex initiator, (2) primary complex stabilizer, and (3) loop destructor—and be complementary to the (free end)/loop-stem-loop tRNA structural element.  相似文献   

6.
One form of aspartic acid tRNA from Drosophila,melanogaster (tRNAAsp) is selectively bound to columns of Con A-Sepharose. Unlike the other Q-containing tRNAs of Drosophila, it therefore appears that tRNAAsp contains the more highly modified nucleoside, Q1 (mannose form) in its anticodon. This is further supported by the chromatographic insensitivity of tRNAAsp to NaIO4 treatment. Utilizing Con A-Sepharose chromatography, tRNAAsp from 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 tRNAAsp.  相似文献   

7.
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. E. coli tRNA1A1a, 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 E. coli tRNA1A1a and confirmation of each of these four predictions.  相似文献   

8.
9.
The effect of codon-anticodon interaction on the structure of two tRNAPhe species was investigated by means of nuclear magnetic resonance spectroscopy. To this end n.m.r.2 spectra of yeast and Escherichia coli tRNAPhe were recorded in the absence and the presence of the oligonucleotides U-U-C-A, U-U-C-G and U-U-C-A-G, which all contain the sequence UUC complementary to the anticodon sequence GAA. The spectra of the hydrogen-bonded protons, the methyl protons and the internucleotide phosphorous nuclei served to monitor the structure of the anticodon loop and of the tRNA in the tRNA-oligonucleotide complex. From the changes in the methyl proton spectra and in the phosphorous spectra it could be concluded that the oligonucleotides bind to the anticodon. Moreover it turned out that the binding constants obtained from these n.m.r. experiments were, within experimental error, equal to the values obtained with other techniques. Using the resonances of the protons hydrogen-bonded between the oligonucleotide and the anticodon loop the structure of the latter could be studied. In particular, binding of the pentanucleotide U-U-C-A-G, which is complementary to the five bases on the 5′ side of the anticodon loop, resulted in the resolution of four to five extra proton resonances indicating that four to five base-pairs are formed between the pentanucleotide and the anticodon loop. The formation of five base-pairs was confirmed by an independent fluorescence binding study. The resonance positions of the hydrogen-bonded protons indicate, that an RNA double helix is formed by the anticodon loop and U-U-C-A-G with the five base-pairs forming a continuous stack. This structure can be accomodated in the so-called 5′ stacked conformation of the anticodon loop, a structure that has been suggested earlier as an alternative to the familiar 3′ stacked conformation in the crystal structure models of yeast tRNAPhe. It turned out that structural adjustments of the anticodon loop to the binding of the oligonucleotides are propagated into the anticodon stem. The relevance of these results with respect to the mechanism of protein synthesis is discussed.  相似文献   

10.
The hexanucleotide Gm-A-A-Y-A-ψp excised from the anticodon loop of yeast tRNAPhe and its constituent oligonucleotides have been studied by ultraviolet absorption spectroscopy, static fluorescence, and circular dichroism. Gm-Ap has a melting point of 45°C and a high melting enthalpy when compared with G-Ap; hence 2′-O-methylation seems to stabilize stacking interactions. The nucleobase Y adjacent to the 3′-side of the anticodon triplet interacts stronger with its 3′-neighboring A than with its 5′-neighboring A. It is concluded that the base Y disconnects the stack of the anticodon itself from the stack of the anticodon stem, thereby setting a reading frame for the mRNA in the course of protein biosynthesis. From the opposite signs of the short-wavelength Cotton effects in the spectra of Gm-A-A-Y-Ap and Gm-A-A-Y, it is concluded that Y after removal of its 3′ neighbor undergoes a dramatic change in its conformation. The fluorescence of the nucleobase Y upon addition of Mg2+ is enhanced in oligonucleotides longer than two. An identical enhancement is observed for tRNAPhe, indicating that this Mg2+ effect is a property of an oligonucleotide segment and does not reflect conformational changes of the whole tRNA. The data presented here reveal that the basic structural features of the anticodon loop are already present in the hexanucleotide Gm-A-A-Y-A-ψp and are not determined by the overall structure of tRNA.  相似文献   

11.
A tRNAPhe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA. As previously reported (Ehrenberg, M., Rigler, R. and Wintermeyer, W. (1979) Biochemistry 18, 4588–4599) in the tRNA derivative the ethidium is present in three states (T1–T3) characterized by different fluorescence decay rates. T-jump experiments show two transitions between the states, a fast one (relaxation time 10–100 ms) between T1 and T2, and a slow one (100–1000 ms) between T2 and T3. In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy. Spermine shifts the distribution of states towards T3, as does Mg2+, but the final ratio [T2][T1] obtained with spermine is higher than with Mg2+, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.  相似文献   

12.
Using singlet-singlet energy transfer, we have measured the distance between the anticodons of two transfer RNAs simultaneously bound to a messengerprogramed Escherichia coli 70 S ribosome. The fluorescent Y base adjacent to the anticodon of yeast tRNAYPhe serves as a donor. A proflavine (Pf) chemically substituted for the Y base in tRNAPfPhe serves as an acceptor. By exploiting the sequential binding properties of 70 S ribosomes for two deacylated tRNAs, we can fill the strong site with either tRNAYPhe or tRNAPfPhe and then the weak site with the other tRNA. In both cases donor quenching and sensitized emission of the acceptor are observed. Analysis of these results leads to an estimate for the Y-proflavine distance of 18 ± 2 Å. This distance is very short and suggests strongly that the two tRNAs are simultaneously in contact with adjacent codons of the message. Separate experiments show that binding of a tRNA to the weak site does not perturb the environment of the hypermodified base of a tRNA bound to the strong site. This supports the assignment of the strong site as the peptidyl site. It also indicates that binding of the second tRNA proceeds without a change in the anticodon structure of a pre-existing tRNA at the peptidyl site.  相似文献   

13.
The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNAPhe. Constructing different anticodon mutants of E. coli tRNAPhe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNAPhe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNAPhe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNAPhe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.  相似文献   

14.
2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNAPhe by tRNA nucleotidyl transferase. tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNAPhe-C-C-3′dA proved to be chargeable.  相似文献   

15.
The enthalpy of the helix-coil conversion of phenylalaninespecific transfer ribonucleic acid from brewer's yeast (tRNAPhebrewer's yeast) has been measured using both an LKB 10700-2 batch miciocalorimeter and an adiabatic differential scanning calorimeter. In the mixing calorimeter the conversion from coil to helix was induced by mixing a tRNAPhe solution with a solution containing an excess of MgSO4. We measured the enthalpy of this reaction stepwise in the temperature range from +9 to +60° C. For the enthalpy of folding of tRNAPhe from coil to helix this method yielded the remarkably high value of ?310 kcalmole of tRNAPhe. With the differential scanning calorimeter in which the helix-coil conversion is simply induced by raising the temperature we found a value of +240 kcalmole of tRNAPhe at a Tm value of 76° C and a value of +200 kcalmole of tRNAPhe at a Tm value of 50° C. A comparison of the apparent van't Hoff enthalpies with the calorimetrically measured enthalpies shows, that the cooperativity of the system increases continually with rising melting temperatures - which are achieved by increasing Mg2+ concentrations - reaching a constant value at about 57° C. Above this temperature value the thermodynamic behaviour of the helix-coil conversion of tRNAPhe may be approximately described by the model of an all-or-none process.  相似文献   

16.
Treatment of E.coli formylmethionine tRNA with sodium bisulfite produces six C → U base changes in the tRNA structure. Four of these modifications have no effect on the ability of tRNAfMet to be aminoacylated or formylated. Prior to bisulfite treatment, Met-tRNAfMet is not able to form a ternary complex with bacterial T factor and GTP, as measured by Sephadex G-50 gel filtration. After bisulfite treatment, a large portion of the modified tRNA is bound as T-GTP-Met-tRNAfMet. Formylation of bisulfite-modified Met-tRNAfMet completely eliminates T factor binding. Unmodified tRNAfMet is unique among the tRNAs sequenced to date in having a non-hydrogen-bonded base at the 5′ terminus. Bisulfite-catalyzed conversion of this unpaired C1 to U1 results in formation of a normal U1-A73 base pair at the end of the acceptor stem. It is likely that this structural alteration is responsible for the recognition of bisulfite-modified Met-tRNAfMet by T factor.  相似文献   

17.
Function of Y in codon-anticodon interaction of tRNA Phe   总被引:7,自引:0,他引:7  
Molar association constants of binding oligonucleotides to the anticodon loops of (yeast) tRNAPhe, (yeast) tRNAHClPhe and (E. coli) tRNAFMet have been determined by equilibrium dialysis. From the temperature dependence of the molar association constants, ΔF, ΔH and ΔS of oligomer-anticodon loop interaction have been determined. The data indicate that the free energy change of codon-anticodon interaction is highly influenced by the presence of a modified purine (tRNAPhe), of an unmodified purine (tRNAFMet) or its absence (tRNAHClPhe). Excision of the modified purine Y in the anticodon loop of tRNAPhe results in a conformational change of the anticodon loop, which is discussed on the basis of the corresponding changes in ΔF, ΔH and ΔS.  相似文献   

18.
Abstract

The anticodon of yeast tRNAAsp, GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNAAsp molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNAPhe. In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNAAsp T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNAPhe. This variation is a consequence of the anticodon-anticodon base pairing which rigidities the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNAAsp substantiate such a correlation.  相似文献   

19.
20.
Two fractions of phenylalanine tRNA (tRNAPhe1 and tRNAPhe2) were purified by BD-cellulose and RPC-5 chromatography of crude tRNA isolated from barley embryos. Successive RPC-5 rechromatography runs of tRNAPhe2 showed its conversion into more stable tRNAPhe1, suggesting that the two fractions have essentially the same primary structure. Both tRNAPhe1 and tRNAPhe2 had about the same acceptor activity, but tRNAPhe2 was aminoacylated much faster than tRNAPhe1. RPC-5 chromatography of crude aminoacylated tRNA showed higher contents of phe-tRNAPhe2 than of phe-tRNAPhe1 but the ratio of these two fractions estimated by relative fluorescence intensity was about 1. Fluorescence spectra of tRNAPhe from barley embryos suggest that it contains Y base similar to Yw from wheat tRNAPhe.  相似文献   

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