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1.
Letters to the editor: Nuclear magnetic resonance studies of protein-RNA interactions. I. The elongation factor Tu-GTP aminoacyl-tRNA complex 总被引:4,自引:0,他引:4
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
2.
The effect of binding of the codon UUC to yeast tRNAPhe was investigated by means of n.m.r.2 spectroscopy and analytical ultracentrifugation. Binding of UUC to the transfer RNA anticodon tends to promote the aggregation of tRNA molecules; this is manifest from a line broadening in the n.m.r. experiments as well as from an increase in s20,w the ultracentrifuge experiments. Such an aggregation of tRNA molecules was not observed upon addition of different oligonucleotides, as described in the accompanying paper. In addition to the general broadening observed in the n.m.r. spectra, specific resonances in the methyl proton spectrum as well as in the hydrogen-bonded proton spectrum are broadened or shifted upon binding of UUC.These results are explained on the basis of the premise that two different tRNA-UUC complexes can exist in solution. It is suggested that the binding of UUC tends to promote a disruption of the m7G46 · m22G22 base-pair and its neighbouring base-pairs.In studying the binding of U-U-U-U to yeast tRNAPhe no resonances of protons hydrogen-bonded between the oligonucleotide and the tRNA could be detected at low temperatures. This indicates, that at these temperatures the lifetime of the tRNA-U-U-U-U complex is substantially shorter than the lifetime of the other tRNA-oligonucleotide complexes studied in this and the accompanying paper under these conditions. 相似文献
3.
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
4.
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
5.
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
6.
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
7.
Interaction of manganese with fragments, complementary fragment recombinations, and whole molecules of yeast phenylalanine specific transfer RNA 总被引:3,自引:0,他引:3
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′-, 3′-, 5′-, 3′-), 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
8.
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
9.
Assignment of the low field proton nuclear magnetic resonance spectrum of yeast phenylalanine transfer RNA to specific base pairs 总被引:12,自引:0,他引:12
D R Lightfoot K L Wong D R Kearns B R Reid R G Shulman 《Journal of molecular biology》1973,78(1):71-89
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 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
10.
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
11.
Complex formation between transfer RNAs with complementary anticodons: use of matrix bound tRNA 总被引:8,自引:0,他引:8
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 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
12.
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
13.
14.
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
15.
The phosphorescence of brewers' yeast phenylalanine transfer RNA has been investigated at 77 °K and at 1.2 °K in pumped liquid helium. Although the phosphorescence at 77 °K originates almost completely from the Y base in the anticodon loop, independent of excitation wavelength, the phosphorescence originates from normal bases with 270 nm excitation at temperatures in the helium range. The low-temperature phosphorescence is assigned to the triplet state of adenosine by optical detection of magnetic resonance measurements. The adenosine phosphorescence at 1.2 °K is quenched by the binding of the codon poly(U), as well as by the removal of Mg2+. The former result indicates that the adenosine phosphorescence originates from the anticodon, -Gm-A-A-, while the second shows that a conformational change introduced by removing Mg2+ (possibly involving unstacking of the anticodon) prevents energy trapping in the anticodon triplet state. The lack of triplet energy transfer from anticodon to Y indicates that Y cannot be stacked with the anticodon in the conformation that is stable at helium temperature. The adenosine phosphorescence of transfer RNAPhe is nearly completely quenched at 77 °K, at least partially due to energy transfer to Y. We think that the thermally activated energy transfer is associated with some mobility of the Y base at 77 °K. Our observations are in contrast with previous results on bakers' yeast tRNAPhe where there is apparently little, if any, energy transfer to Y from the normal nucleotides at 80 °K with 265 nm excitation. Optically detected magnetic resonance measurements on the triplet state of Y base in various environments indicate that removal of Mg2+ causes a shift of the Y base in tRNAPhe to a more solvent-exposed position, whereas the binding of poly(U) has little effect on the environment of Y. 相似文献
16.
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
17.
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
18.
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
19.
Yu. N. Vorobjev 《Molecular Biology》2005,39(5):777-784
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
20.
Localization of Two Recognition Sites in Yeast Valine tRNA I 总被引:7,自引:0,他引:7
AS a part of our research on the structure–function relationships of tRNAvalI we have been mapping the regions that take part in the recognition of valyl tRNA ligase. Using the “dissected molecule” method1, we have shown that associated molecules consisting of tRNAValI fragments lacking nucleotides in the anticodon loop, the dihydrouridine loop (D) or the thymidine loop (T) retain their acceptor activity. By contrast, dissected molecules devoid of the pentanucleotide A36CACGp (the sequence A36C belongs to the anticodon T35AC) or lacking any quarter (F1–19, F17–35 or F36–57) are inactive2–4. Here we report a study of the acceptor activity of other incomplete tRNAvalI molecules. The principal inference is that the dinucleotides A36Cp in the anticodon loop and 5′-terminal pG1Gp in the CCA stem are at least parts of two different recognition sites of this tRNA. 相似文献