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1.
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   

2.
tRNAPhe was isolated from posterior silkgland from Bombyx mori and hydrolysed to mixtures of oligonucleotides. |32P|5′ end labelling of the oligonucleotides and sequence study indicates that the major component of Bombyx mori tRNAPhe is similar to mammalian tRNAPhe, the minor component differing from the major one by one nucleotide only.  相似文献   

3.
Abstract

NMR spectroscopy was used to determine the solution structures of RNA oligonucleotides comprising the anticodon domain of tRNALys,3. The structural effects of the pseudouridine modification at position 39 were investigated and are well correlated with changes in thermodynamic parameters. The loop conformation differs from that seen in tRNAPhe and provides an explanation of the critical role of modification in this tRNA.  相似文献   

4.
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   

5.
6.
The anticodon-anticodon complex   总被引:6,自引:0,他引:6  
Gel electrophoresis has been used to measure the binding between two tRNAs with complementary anticodons, tRNAVal (Escherichia coli) (anticodon X,A,C) and tRNATyr (E. coli) (anticodon Q,U,A). The association constant K at 0 °C was found to be 4 × 105 m?1 which is about three orders of magnitude greater than the association constant for tRNATyr (E. coli) binding its trinucleotide codon UAC. The temperature dependence of K suggests that this results from the rigidity of the anticodon loop. tRNATyr (E. coli) binds an order of magnitude more weakly to tRNAVal (yeast) than to tRNAVal (E. coli), presumably because it contains the wobble base pair A · I. The relationship between the anticodon-anticodon complex and codon recognition is discussed.  相似文献   

7.
8.
9.
The major species of valine specific tRNA was isolated from human placenta, degraded to oligonucleotides, and shown to have the nucleotide sequence pG-U-U-U-C-C-G-U-A-G-U-G-U?-A-G-D-G-G-D-D-A-U-C-A-C-m2G-U?-U-C-G-C-C-U-(I or C)-A-C-A-C-G-C-G-A-A-A-G-m7G-D-m5C-m5C-C-C-G-G-U-U?-C-G-m1A-A-A-C-C-G-G-G-C-G-G-A-A-A-C-A-C-C-AOH. This human placental tRNAVal differs from the major species of mouse myeloma tRNAVal only in that it contains either I or C in the wobble position of the anticodon, and totally lacks 2′-O-methylcytosine and 5-methylcytosine in the anticodon loop.  相似文献   

10.
11.
Bacterial translation initiation factor 2 (IF2) is a GTPase that promotes the binding of the initiator fMet‐tRNAfMet to the 30S ribosomal subunit. It is often assumed that IF2 delivers fMet‐tRNAfMet to the ribosome in a ternary complex, IF2·GTP·fMet‐tRNAfMet. By using rapid kinetic techniques, we show here that binding of IF2·GTP to the 30S ribosomal subunit precedes and is independent of fMet‐tRNAfMet binding. The ternary complex formed in solution by IF2·GTP and fMet‐tRNA is unstable and dissociates before IF2·GTP and, subsequently, fMet‐tRNAfMet bind to the 30S subunit. Ribosome‐bound IF2 might accelerate the recruitment of fMet‐tRNAfMet to the 30S initiation complex by providing anchoring interactions or inducing a favourable ribosome conformation. The mechanism of action of IF2 seems to be different from that of tRNA carriers such as EF‐Tu, SelB and eukaryotic initiation factor 2 (eIF2), instead resembling that of eIF5B, the eukaryotic subunit association factor.  相似文献   

12.
Abstract

The existence of specific sites in tRNA for the binding of divalent cations has been seriously questioned by electrostatic considerations [Leroy & Guéron (1979) Biopolymers, 16, 2429–2446], However, our earlier studies of the binding of Mg2+ and Mn2+ to yeast tRNATyr have indicated that spermine creates new binding sites for divalent cations [Weygand-Durasevi? et al. (1977) Biochim. Biophys. Acta, 479, 332–344; Nöthig-Laslo et al. (1981) Eur. J. Biochem. 117, 263–267]. We have now used yeast tRNATyr, spin labeled at the hypermodified purine (i6A-37) in the anticodon loop, to study the effect of spermine on the binding of manganese ions. The presence of eight spermine molecules per tRNATyr at high ionic strength (0.2 M NaCl, 0.05 M triethanolamine-HCl) and at low temperature (7°C) enhances the binding of manganese to tRNATyr. This effect could not be explained by electrostatic binding. The initial binding of manganese to tRNATyr affects the motional properties of the spin label indicating a change of the conformation of the anticodon loop. From the absence of the paramagnetic effect of manganese on the ESR spectra of the spin label one can conclude that the first binding site for manganese is at a distance from i6A-37, influencing the spin label motion through a long-range effect. The enhancement of the binding of manganese to tRNATyr by spermine is lost upon destruction of its specific macromolecular structure and it does not occur in single stranded or in double-stranded polynucleotides. The observed effect can be explained by the binding of Mn2+ to new sites, created by the binding of spermine, which are specific for the macromolecular structure of tRNA.  相似文献   

13.
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   

14.
A new chemical method for radioactive labeling of single-stranded regions of RNA has been used to probe the three-dimensional structure of E. coli tRNAfMet in solution. The procedure involves conversion of cytosine residues to N4-[14C]methylcytosines by treatment with 14CH3NH2 and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces 14C-labeled oligonucleotides which comigrate with the corresponding unlabeled oligonucleotides, facilitating structural analysis. By this procedure, E. coli tRNAfMet has been found to contain only six reactive cytosines: C1, C16, C17, C35, C75 and C76. In addition, slow reaction at Cm33 was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNAfMet obtained by two other chemical methods. The methylamine-bisulfite procedure is recommended for studying the ordered structure of more complex polyribonucleotides such as viral and ribosomal RNAs.  相似文献   

15.
THE degree of degeneracy of the genetic code varies for the twenty amino-acids: between one and six different triplets are assigned to a single amino-acid. Four triplets GUU, GUC, GUA, GUG code for the amino-acid valine1,2. Two valine specific tRNAs have been separated by fractionation of mixed E. coli tRNA3; tRNAval1 is specific for GUAG and tRNAval2 corresponds to GUUC (see also ref. 1 for binding properties). Recent studies showed that although both species are recognized by the single activating enzyme present in E. coli, the association constant (Ka) for the minor species, tRNAval2 (?20% of total acceptor), is an order of magnitude higher than the association constant of the major species, tRNAval 41. As a first step to comparing the structures of these two tRNAs, we analysed the base sequences of the major and minor species. We recently published the nucleotide sequence of tRNAval 51; we report here the sequence of two minor subspecies (quite similar to each other) that comprise the tRNAval2 acceptor and we comment on the significance of the sequence homologies in relation to the problems of enzyme recognition and tRNA evolution.  相似文献   

16.
The N-terminally myristoylated matrix (MA) domain of the HIV-1 Gag polyprotein promotes virus assembly by targeting Gag to the inner leaflet of the plasma membrane. Recent studies indicate that, prior to membrane binding, MA associates with cytoplasmic tRNAs (including tRNALys3), and in vitro studies of tRNA-dependent MA interactions with model membranes have led to proposals that competitive tRNA interactions contribute to membrane discrimination. We have characterized interactions between native, mutant, and unmyristylated (myr-) MA proteins and recombinant tRNALys3 by NMR spectroscopy and isothermal titration calorimetry. NMR experiments confirm that tRNALys3 interacts with a patch of basic residues that are also important for binding to the plasma membrane marker, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Unexpectedly, the affinity of MA for tRNALys3 (Kd = 0.63 ± 0.03 μM) is approximately 1 order of magnitude greater than its affinity for PI(4,5)P2-enriched liposomes (Kd(apparent) = 10.2 ± 2.1 μM), and NMR studies indicate that tRNALys3 binding blocks MA association with liposomes, including those enriched with PI(4,5)P2, phosphatidylserine, and cholesterol. However, the affinity of MA for tRNALys3 is diminished by mutations or sample conditions that promote myristate exposure. Since Gag–Gag interactions are known to promote myristate exposure, our findings support virus assembly models in which membrane targeting and genome binding are mechanistically coupled.  相似文献   

17.
Transfer RNA from Escherichia coli C6, a Met, Cys, relA mutant, was previously shown to contain an altered tRNAIle which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676–7683). We now report the purification of this altered tRNAIle and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNAIle purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNAIle (from cysteine-starved cells) was found to have a 5-fold increased Vmax in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl ∼ AMP · Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNAIle bound more efficiently to its synthetase compared to normal tRNAIle. Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNAIle contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNAIle contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNAIle without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNAIle has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNAIle from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNAIle for aminoacylation is discussed.  相似文献   

18.
Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm5U34), 5-methoxycarbonylmethyluridine (mcm5U34), and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNALys s2 UUU, tRNAGln s2 UUG, and tRNAGlu s2 UUC, which in a wild-type background contain the mcm5s2U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U34. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm5s2U nucleoside in tRNALys mcm5s2UUU, tRNAGln mcm5s2UUG, and tRNAGlu mcm5s2UUC. These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U34 are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.  相似文献   

19.
Abstract

The synthesis and the enzymatic studies of modified oligonucleotides containing a PNA modified PNA-DNA dimer block and a new acyclic racemic serinol nucleoside is described. We show that both, the PNA-DNA dimer block1 and the modified PNA-spacer (acyclic serinol nucleoside)2 can be used as modified templates for the enzymatic generation of single stranded DNA. Degradation studies of the oligonucleotides containing the PNA-DNA dimer block with snake venom phosphodiesterase show that the modified oligonucleotides are stable towards exonucleolytic degradation.  相似文献   

20.
Plasma-membrane-specific localization of Gag, an essential step in HIV-1 particle assembly, is regulated by the interaction of the Gag MA domain with PI(4,5)P2 and tRNA-mediated inhibition of non-specific or premature membrane binding. Different tRNAs inhibit PI(4,5)P2-independent membrane binding to varying degrees in vitro; however, the structural determinants for this difference remain unknown. Here we demonstrate that membrane binding of full-length Gag synthesized in vitro using reticulocyte lysates is inhibited when RNAs that contain the anticodon arm of tRNAPro, but not that of tRNALys3, are added exogenously. In contrast, in the context of a liposome binding assay in which the effects of tRNAs on purified MA were tested, full-length tRNALys3 showed greater inhibition of MA membrane binding than full-length tRNAPro. While transplantation of the D loop sequence of tRNALys3 into tRNAPro resulted in a modest increase in the inhibitory effect relative to WT tRNAPro, replacing the entire D arm sequence with that of tRNALys3 was necessary to confer the full inhibitory effects upon tRNAPro. Together, these results demonstrate that the D arm of tRNALys3 is a major determinant of strong inhibition of MA membrane binding and that this inhibitory effect requires not only the D loop, which was recently reported to contact the MA highly basic region, but the loop sequence in the context of the D arm structure.  相似文献   

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