共查询到20条相似文献,搜索用时 62 毫秒
1.
Alfred Maelicke Friedrich Von Der Haar Mathias Sprinzl Friedrich Cramer 《Biopolymers》1975,14(1):155-171
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
2.
Kazuyuki Nagamatsu 《Journal of biomolecular structure & dynamics》2013,31(4):729-739
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.
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
4.
High resolution NMR study of the melting of yeast tRNA Phe 总被引:7,自引:0,他引:7
C W Hilbers R G Shulman S H Kim 《Biochemical and biophysical research communications》1973,55(3):953-960
The 300 MHz NMR spectra of the hydrogen bonded NH ring protons of tRNAYeastPhe have been measured as a function of temperature. In the presence of Mg++ two resonances, one from the Aψ base pair and the other probably from the neighboring base pair, disappear between 56 and 58°C. In the absence of Mg++ the DHU stem, the acceptor stem (in particular its AU base pair #6 and #7) and the Aψ base pair in the anticodon stem melt slightly earlier than the other parts of the molecule. Since the DHU stems in tRNAYeastPhe and tRNAColifMet have the same base pairing scheme it is interesting that their melting behavior is entirely different in both molecules. This is discussed in terms of the tertiary structure. 相似文献
5.
When yeast tRNAPfPhe, a derivative of tRNAPhe in which proflavine replaces the Y base, is bound simultaneously to both the peptidyl and aminoacyl sites of a 70 S Escherichia coli ribosome, there is a rapid mutual energy transfer between the two bound tRNAs. Analysis of this energy transfer yields an upper limit for the proflavine-proflavine distance of 20 Å. It also allows an unequivocal measurement of the emission spectrum of tRNAPfPhe bound at the aminoacyl site. In the presence of message this spectrum is very different from that seen in the peptidyl site, implying that in the two sites the hypermodified bases exist in significantly different environments. The rapid energy transfer leads to some loss of fluorescence anisotropy. This can be analyzed to obtain an estimate of the angle between the two proflavines: 28 ° ± 10 ° or 152 ° ± 10 °. Taken together all of these results place severe constraints on possible models of codon-anticodon complexes. The mutual energy transfer seen and analyzed on the ribosome is a convenient aspect of fluorescence spectroscopy, and it is one that should see broad application where multiple copies of a fluorescent ligand interact on a macromolecular substrate. 相似文献
6.
Soboleva N. G. Makhno V. I. Konevega A. L. Semenkov Yu. P. Katunin V. I. 《Molecular Biology》2003,37(1):110-115
To estimate the effect of modified nucleotide 37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNAPhe
+Y and Phe-tRNAPhe
–Y) with the A site of complex [70S · poly(U) · deacylated tRNAPhe in the P site] was assayed at 0–20°C. As comparisons with native Phe-tRNAPhe
+Y showed, removal of the Y base decreased the association constant of Phe-tRNAPhe
–Y and the complex by an order of magnitude at every temperature tested, and increased the enthalpy of their interaction by 23 kJ/mol. When the Y base was present in the anticodon loop of deacylated tRNAPhe bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNAPhe
–Y but not for Phe-tRNAPhe
+Y. Thus, the modified nucleotide 3" of the Phe-tRNAPhe anticodon stabilized the codon–anticodon interaction both in the A and P sites of the 70S ribosome. 相似文献
7.
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
8.
The structure of yeast transfer RNA aspartic acid has been refined in one crystal form to 3 Å resolution using the restrained least-squares method of Hendrickson and Konnert and real-space fitting using the FRODO program of Jones. The final Crystallographic discrepancy index R is 23.5% for 4585 reflections with magnitudes twice their standard deviations between 10 and 3 Å. With lower occupancies for some residues of the D-loop, the phosphate U1, and the base U33, the R-factor is 22.3%. The adaptation of the restrained least-squares program for nucleic acids and the progress of the refinement are described. The conformations are analysed with respect to stereochemistry and folding of the backbone. The contacts and hydrogen bonds of the secondary structure are compared with those of yeast tRNAPhe. The presence of only four bases in the variable loop, instead of five as in yeast tRNAPhe, leads to a rotation of residue 48 and a lateral movement of residue 46. These two rearrangements induce different environments for [U8 … A14] … A21 as well as for A9 and G45. Otherwise, all tertiary contacts observed in yeast tRNAPhe are present in yeast tRNAAsp, except for the absence of hydrogen-bonding between G18 of the D-loop and C56 of the T-loop. The presence of anticodon triplet pairing leads to a distribution of temperature factors different from that observed in yeast tRNAPhe with a stabilization of the AC stem-and-loop and a destabilization of the T and D-loops. We are inclined to suggest that the labilization of the interactions between the T and D-loops is a consequence of the interaction of the anticodon triplets of symmetry-related molecules through hydrogen bonding, which mimics the interaction between the anticodon and its cognate codon on the messenger RNA. 相似文献
9.
Spectroscopic properties of oligonucleotides excised from the anticodon region of phenylalanine tRNA from yeast 总被引:6,自引:0,他引:6
Ultraviolet absorption and static fluorescence properties of hexanucleotide (Gm-A-A-Y-A-ψp) and a dodecanucleotide (A-Cm-U-Gm-A-A-Y-A-ψ-m5C-U-Gp) excised from the anticodon region of phenylalanine tRNA from yeast have been studied with respect to temperature, pH, ionic strength, and Mg2+ concentration. At low temperature these oligomers have a largely stacked structure. Only the melting data of the dodecanucleotide in absence of Mg2+ fit a two-state model. From the different melting behavior of the oligonucleotides after excision of base Y, a rodlike structure of the hexanucleotide produced by stacking interactions can be concluded. The Y fluorescence increase produced by Mg2+ has been used to evaluate the binding equilibria between Mg2+ and the oligonucleotides. One strong binding site per oligonucleotide and a greater number of weak binding sites have been found. The fluorescence of the free base Y is not influenced by Mg2+. The dodecanucleotide enhances the ethidium fluorescence to the same extent as tRNAPhe and produces comparable shifts in the excitation and emission spectra. Therefore a double helical structure for this oligomer under the assay conditions is suggested. Only weak binding of ethidium to the hexanucleotide is observed, indicating that intercalation of the dye into its structure is not favored. The data show the decisive role of the nucleobase Y in maintaining a rigid stacked structure of the anticodon nucleotides. This structure is stabilized by high ionic strength, Mg2+, and ethidium. 相似文献
10.
Trans-dichlorodiammineplatinum (II) reacts with yeast phenylalanine transfer RNA to yield a major platinum binding site. The tightly bound platinum has been located on the oligonucleotide Gm-A-A-Y-A-ψp containing the anticodon by standard fingerprinting methods using 32P-labelled tRNAPhe. This site corresponds to a single major platinum site identified during an X-ray crystallographic analysis of yeast tRNAPhe. The solution studies have given confidence to the assignment of part of the 3 Å electron density map to the anticodon region of the molecular structure of yeast tRNAPhe. 相似文献
11.
D. Moras A. C. Dock P. Dumas E. Westhof P. Romby J. P. Ebel 《Journal of biomolecular structure & dynamics》2013,31(3):479-493
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
12.
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 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 of tRNAPhe at a Tm value of 76° C and a value of +200 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
13.
Damian Labuda Henri Grosjean George Striker Dietmar Pörschke 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,698(3):230-236
In order to learn about the effect of the G:U wobble interaction we characterized the codon:anticodon binding between triplets: UUC, UUU and yeast tRNAPhe (anticodon GmAA) as well as the anticodon:anticodon binding between Escherichia coli tRNAGlu2, E. coli tRNALys (anticodons: mam5s2UUC, and mam5s2UUU, respectively) and tRNAPhe from yeast and E. coli (anticodon GAA) using equilibrium fluorescence titrations and temperature jump measurements with fluorescence and absorption detection. The difference in stability constants between complexes involving a G:U pair rather than a usual G:C basepair is in the range of one order of magnitude and is mainly due to the shorter lifetime of the complex involving G:U in the wobble position. This difference is more pronounced when the codon triplet is structured, i.e., is built in the anticodon loop of a tRNA. The reaction enthalpies of the anticodon:anticodon complexes involving G:U mismatching were found to be about 4 kcal/mol smaller, and the melting temperatures more than 20°C lower, than those of the corresponding complexes with the G:C basepair. The results are discussed in terms of different strategies that might be used in the cell in order to minimize the effect of different lifetimes of codon-tRNA complexes. Differences in these lifetimes may be used for the modulation of the translation efficiency. 相似文献
14.
15.
On the triplet states of polynucleotide--acridine complexes. I. Triplet energy delocalization in the 9-aminoacridine-DNA complex 总被引:2,自引:0,他引:2
W C Galley 《Biopolymers》1968,6(9):1279-1296
Phosphorescence and fluorescence from the dye in complexes of DNA with 9-amino-acridine and acridine orange in a glycerol-H2O glass have been measured at 77°K. The dependence of the p/fratio for 9-aminoacridine on the exciting wavelength demonstrates triplet–triplet energy transfer from DNA to dye. The result provides evidence for π electron overlap between the dye and the bases of native DNA. The observation that the magnitude of the enhancement in ultraviolet-excited dye phosphorescence increases with the base to dye ratio indicates triplet delocalization in the polymer. Preliminary flash experiments provide evidence that this delocalization is not limited by slow diffusion of the triplet exciton. The inability to detect transfer on denaturation of the DNA illustrates the sensitivity of triplet–triplet energy transfer to the conformation of the macromolecular complex. 相似文献
16.
Codon-Anticodon Binding in tRNA<Superscript>phe</Superscript> 总被引:5,自引:0,他引:5
THE anticodon loop of tRNAphe of baker's yeast has the sequence (5′ to 3′) AY A A MeG U MeC. The unusual base Y, adjacent to the anticodon (AA MeG), is the only nucleotide in this tRNA which fluoresces at room temperature and because it absorbs to the red of all other bases, the excitation energy is localized on it exclusively. (7-Methyl guanine is another base in tRNAphe which fluoresces in these conditions but its emission is so weak that it can only be observed in tRNAphe from which Y has been excised.) The fluorescence spectrum undergoes a small blue shift in the presence of the complementary codon1 and we report now the use of this shift to determine the association constants for this binding at several temperatures. The results suggest a simple thermodynamic model for the codon recognition step during protein synthesis. 相似文献
17.
Conformational change in yeast tRNAAsp 总被引:1,自引:0,他引:1
The structure of yeast tRNAAsp in aqueous solutions has been analyzed in the light of results obtained from Raman spectra recorded at from 5 to 82°C and compared to those of tRNAPhe. Firm evidence is given of a reversible conformation transition for tRNAAsp at 20°C. This transition is observed for the first time in the tRNA series. The low-temperature conformation appears to have a more regular ribose–phosphate backbone and a more effective G base-stacking. This conformational change, which occurs essentially in the D loop, could be connected to the existence of two (A and B) crystal forms obtained depending on crystallization conditions. The melting temperatures, which are different for each base stacking in tRNAAsp, lie in a range of about 70°C, much higher than for tRNAPhe. This fact is interpreted by a higher ratio of G-C base pairs in tRNAAsp. 相似文献
18.
19.
The high-resolution (300 MHz) proton nmr spectrum of E. coli tRNAfMet has been examined in 0.17M NaCl, with and without Mg2+, and at various temperatures. In light of recent studies of other E. coli tRNA and fragments of tRNAfMet, some low field (11–15 ppm) resonances previously assigned to secondary structure base pairs are reassigned to a tertiary structure A14–S4U8 base pair and a protected uridine residue in the anticodon loop. These two resonances and other low field resonances which are assigned to secondary structure base pairs are used to monitor the thermal unfolding of the molecule. In the absence of Mg2+ the tertiary structure base pair is present only to ~45°C, but in the presence of Mg2+ it remains until at least 70°C. Analysis of the temperature dependence of other low field resonances indicates that the melting of the dihydrouridine stem occurs more or less simultaneously with the loss of tertiary structure. The observation of the resonance from the A14–S4U8 base pair proves that tertiary structure is present in this molecule below 40°C, even in the absence of Mg2+. 相似文献
20.
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