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1.
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   

2.
The effect of aminoacylation and ternary complex formation with elongation factor Tu•GTP on the tertiary structure of yeast tRNAPhe was examined by 1H-NMR spectroscopy. Esterification of phenylalanine to tRNAPhe does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA. Complex formation of Phe-tRNAPhe with elongation factor Tu•GTP results in a broadening of all imino proton resonances of the tRNA. The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNAPhe upon binding to elongation factor Tu•GTP. All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNAPhe•elongation factor Tu•GTP ternary complex. Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNAPhe preserves its typical L-shaped tertiary structure in the complex. At high (> 10−4 M) ligand concentrations a complex between tRNAPhe and elongation factor Tu•GDP can be detected on the NMR time-scale. Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure. Using the known tertiary structures of yeast tRNAPhe and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.  相似文献   

3.
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   

4.
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   

5.
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   

6.
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   

7.
Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and TΨC stems of Saccharomyces cerevisiae tRNAPhe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNACAGLeu resulted in similar changes in binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNAPhe resulted in chimeras with EF-Tu binding affinities similar to those for the donor tRNA. Finally, analysis of double- and triple-base-pair mutants of tRNAPhe showed that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the TΨC stem primarily account for tRNA binding specificity to EF-Tu.  相似文献   

8.
9.
The activity of tRNA methyltransferases present in the cerebellum of 6- and 21-day-old nonicteric and icteric Gunn rats was compared using purifiedE. coli tRNAs as substrates. At 6 days the tRNA methyltransferases of the icteric animals were significantly more effective in methylating tRNAGlu 2 and tRNAPhe than were those of their nonicteric counterparts. This relationship reversed itself at 21 days. The action of the tRNA methyltransferases from the 6-day-old icteric animals led to higher proportions of 1-methyladenine in tRNAGlu 2 and tRNAPhe than were obtained using the corresponding enzymes of the nonicteric animals. The proportion ofN 2-methylguanine was also higher, yet only in tRNAfMet and not in tRNAPhe. The study reveals much more extensive fluctuations in the activity and in the substrate recognition specificity among the cerebellar tRNA methyltransferases of the icteric than among those of the nonicteric controls during the crucial 6–21 day period of cerebellar development.  相似文献   

10.
Conformational transitions in several individual tRNAs (tRNA inff supMet , tRNAPhe from E. coli, tRNA inf1 supVal , tRNASer, tRNAPhe from yeast) have been studied under various environmental conditions. The binding isotherms studies for dyes-tRNA complexes exhibited similarities in conformational states of all tRNAs investigated at low ionic strength (0.01 M NaCl). By contrast, at high ionic strength (0.4 M NaCl or 2×10-4 M Mg2+) a marked difference is found in structural features of tRNA inff supMet as compared with other tRNAs used. The tRNA inff supMet is the only tRNA species that does not reveal the strong type of complexes with ethidium bromide, acriflavine and acridine orange.  相似文献   

11.
Summary Eight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNAVal(GAC), tRNAAsn(GUU), tRNAArg(ACG), tRNALeu(CAA), tRNATyr(GUA), tRNAGlu(UUC), tRNAHis(GUG), and tRNAArg(UCU) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition,two other tRNA genes, tRNAGly (UCC) and tRNAIle(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90–100% homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5 flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNAGly(UCC), and the tRNAGlu(UUC) contains GATTC in its T-loop.  相似文献   

12.
For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of –TΔSb, and by the negative value of ΔCp. On the other hand, the large negative enthalpy is the dominant contribution to ΔGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant K d is decreased 50-fold in the presence of tRNAGlu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3’-OH oxygen of the 3’-terminal ribose of tRNAGlu in the Glu-AMS•GluRS•tRNAGlu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNAGlu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.  相似文献   

13.
The uranyl(VI) ion, UO, cleaves yeast tRNAPhe both thermally and photochemically. Photochemical cleavage takes place at all positions but exhibits maxima at G10, G18, G30, A38, C49 and A62. Furthermore, in the presence of stoichiometric concentrations of citrate, the cleavage is generally suppressed except that strong cleavage at positions G10 and C48–U50 persists, indicating the presence of a high-affinity metal-ion binding site. It is proposed that these photocleavage sites reflect the tertiary structure of the yeast tRNAPhe molecule in terms of D-loop/T-loop interaction and anticodon loop conformation and that uranyl-mediated photocleavage of RNA may be used as a probe of RNA tertiary structure, and in particular for identifying binding sites for divalent metal ions. Thus a high-affinity metal-ion binding site is inferred in the Rcentral pocket' formed by the D-loop, the T-loop and the acceptor stem.  相似文献   

14.
O W Odom  B B Craig  B A Hardesty 《Biopolymers》1978,17(12):2909-2931
The Y-base of yeast tRNAPhe was replaced by the fluorophores 1-aminoanthracene or proflavine to yield derivatives which are active in all of the reactions of peptide elongation on reticulocyte ribosomes. The relatively long lifetime, higher quantum yield, and environmental sensitivity of 1-aminoanthracene make it a particulary useful adjunct to the Y-base in studying conformational changes in the anticodon region. The absorption and emission spectra of 1-aminoanthracene in tRNA in solutions in which it is active in peptide synthesis indicate that the probe is in a hydrophobic environment, apparently provided by stacking with the adjacent bases in the anticodon loop. The proflavine derivative, tRNA, was employed in iodide quenching, D2O enhancement, and fluorescence depolarization experiments. The results indicate that the fluorophore in partially but not completely protected from the solvent. Anisotropy studies indicate that in solutions approximating those which support peptide synthesis on ribosomes, the probes have significant but restricted flexibility within the anticodon loop. Considered with nmr data and Y-base fluorescence from crystals of tRNA, the results indicate that the solution and crystal structures of tRNAPhe are very similar. In turn, fluorescene from modified tRNAPhe bound to ribosomes is similar to that observed in solution. It is of special significance for future experiments involving nonradiative energy transfer that these probles adjacent to the anticodon retain independent flexibility when bound to ribosomes with poly(U). The tRNAPhe itself appears to be held rigidly on the ribosomes. It is concluded that within the limits dictated by the position and sensitivity of the probes used in this study, the mechanism of tRNAPhe binding to ribosomes and the movement of tRNA and mRNA during the translocation steps of peptide synthesis can be interpreted in terms of the well-defined crystal structure of tRNAPhe.  相似文献   

15.
The 300 MHz high-resolution nuclear magnetic resonance spectra of hydrogen-bonded protons in Escherichia coli tRNAGlu and yeast tRNAPhe have previously been reported and the resolved resonances assigned to specific base-pairs. Here we show that in complexes of these two tRNAs with elongation factor Tu there is no discernible loss of base-paired protons. Within the experimental accuracy this means that no helical arms open upon complex formation.  相似文献   

16.
It is shown that yeast tRNAPhe, chemically coupled by its oxidized 3′CpCpA end behaves exactly as free tRNAPhe in its ability to form a specific complex with E. coli tRNA2Glu having a complementary anticodon. The results support models of tRNA in which the 3′CpCpAOH end and the anticodon are not closely associated in the tertiary structure, and provide a convenient tool of general use to characterize others pairs of tRNA having complementary anticodons, as well as for highly selective purification of certain tRNA species.  相似文献   

17.
An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N2-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m2G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNAArg, tRNAPhe, and tRNAVal yielded significantly more m2G than the bulk tRNA. The Km for tRNAArg in the methylation reaction with enzymes from either tissue was 7.8 × 10−7 M as compared to the value 1 × 10−5 M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNAArg, tRNAPhe, and tRNAVal, A-m2G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5′-end contained in a sequence (s4)U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.  相似文献   

18.
19.
High-resolution proton nuclear magnetic resonance spectra at 220 and 300 MHz have been used to investigate the base-pairing structure of fragments of yeast tRNAPhe, of chemically modified tRNAPhe and of intact tRNAPhe. To a very good approximation the positions of the fragment spectra are additive within 0·2 part per million, indicating that factors responsible for certain structural features in the intact molecule are already present in the smaller fragments (half molecules, hairpins and 34 molecules). A simple first-order ring-current shift theory taken in conjunction with the cloverleaf model for tRNAPhe (RajBhandary et al., 1967) has been used to predict the low-field (? 15 to ?11 part per million) nuclear magnetic resonance spectra and make assignments of the resolved resonances to ring NH protons of specific base pairs. The general agreement between the predicted and observed spectra to within 0·2 part per million confirms in detail the cloverleaf model for the secondary structure of tRNAPhe in solution. It is also established that ring-current shifts are the principal factor responsible for the wide range of shifts observed in the low-field spectra. As a result it is evident that the resonances are very sensitive to small changes in the secondary structure and in some cases changes in the interbase distance as small as 0·2 Å could easily be detected. It is also clear from the analysis that certain of the resonances are sensitive to the tertiary structure of the molecule and specific examples are discussed. As with our previous study, we find no evidence for any strong Watson-Crick type base pairs beyond those predicted by the cloverleaf structure.  相似文献   

20.
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