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R Rigler

Publications and source records attributed to R Rigler.

At least 91 records · Page 5Linked to original sources
PubMed1984

Separation of translational and rotational contributions in solution studies using fluorescence photobleaching recovery.

Although fluorescence photobleaching recovery (FPR) experiments are usually interpreted in terms of the translational motions of a fluorescently labeled species, rotational motions can also modulate recovery through the cosine-squared laws for dipolar absorption and emission processes. In a complex interacting system, translational and rotational contributions may both be simultaneously present. We show how these contributions can be separated in solution studies using an FPR setup in which (a) the linear polarization of the low-intensity observation beam and the high-intensity photobleaching pulse can be varied independently, and (b) all emitted fluorescent photons are counted equally. The fluorescence recovery signal obtained with the observation beam polarized at the magic angle, 54.7 degrees, from the bleach polarization direction is independent of label orientation, whereas the anisotropy function formed from a combination of parallel and perpendicular polarizations isolates the orientational recovery. The anisotropy function is identical to that in fluorescence correlation spectroscopy and, for rigid-body rotational diffusion, can be expressed as a sum of five exponential terms.

Mathematics↗
PubMed1983

The influence of spermine on the structural dynamics of yeast tRNAPhe.

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.

Kinetics↗
PubMed1983

Dynamics of tRNA.

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Amino Acyl-tRNA Synthetases↗
PubMed1982

Cross-correlation laser scattering.

Cross-correlation between two detectors was applied to analyze laser light-scattering fluctuations. Laser scattering from random concentration fluctuations is spatially coherent over small angular areas that are inversely proportional in size to the dimension of the scattering volume. By cross-correlating scattering intensity fluctuations in different angles, the correlation due to relaxation of concentration fluctuations is practically eliminated, and correlations reflecting changes in the scattering from the individual particles can be enhanced. Rotational diffusion of assymetric particles, conformational relaxation of random coils, and association-dissociation dynamics are determined here using the above approach.

DNA, Bacterial↗
PubMed1982

Structural variability of tRNA: small-angle x-ray scattering of the yeast tRNAphe-Escherichia coli tRNAGlu2 complex.

The structure of the complex formed in solution between yeast tRNAPhe and Escherichia coli tRNAGlu2 has been studied by small-angle x-ray scattering. The complex has a radius of gyration of 4.0 nm and an electron-pair distance distribution that is incompatible with a model composed to two tRNAs joined at their complementary anticodons and exhibiting the L shape seen in the crystal. Instead a model in which the two tRNAs, still bound via the anticodons, assume a conformation with the acceptor arms folded toward the anticodon arms agrees with the observed scattering curves.

Anticodon↗
PubMed1981

Conformational states of yeast tRNA Phe in the complex with cognate and non cognate synthetases.

The influence of phenylalanyl-tRNA synthetase and seryl-tRNA synthetase on the conformation and structural kinetics of yeast tRNA Phe was investigated. Ethidium substituted for dihydrouracil at position 16 or 17 was used as a structural probe, showing the existence of three conformational states in tRNA. The distribution of states (T1, T2, T3) is changed only by the cognate synthetase towards T3 which probably is related to the X-ray structure. The binding of phenylalanyl-tRNA synthetase leads to an about 10-fold increase in the fast transition T1 in equilibrium or formed from T2 which has been assigned to changes in the anticodon loop conformation and to a 2-3 fold increase in the slow transition which probably extends to other parts of the tRNA molecule. The observed rates for the transition T2 in equilibrium or formed from T3 are close to that observed for the transfer of the activated phenylalanine to tRNA Phe. This raises the possibility that the conformational transition in tRNA is the rate limiting step in the charging reaction.

Amino Acyl-tRNA Synthetases↗
PubMed1980

Fluorescence relaxation of proflavin-deoxyribonucleic acid interaction. Kinetic properties of a base-specific reaction.

The kinetics of proflavin binding to Micrococcus lysodeicticus deoxyribonucleic acid (DNA [(G-C) content 72%], to Bacillus megaterium DNA [(A-T) content 70%], and to the polydeoxyribonucleotides poly[d(G-C)] and poly[d(A-T)] was studied with fluorescence temperature-jump methods. Poly[d(A-T)] binds proflavin in a two-step reaction with a preequilibrium. Poly[d(G-C)] is characterized by a bimolecular reaction. The binding of acridines to natural DNAs is shown to be characterized by different types of sites whose properties depend on the base composition. The sites have considerable enthalpic differences which result in exchange of dye molecules between them when the temperature is changed. Also, on natural DNAs A-T base pairs are associated with a rapidly equilibrating external complex which is absent or much weaker for G-C base pairs.

Acridines↗
PubMed1980

Characterisation of a new, fully active fluorescent derivative of E. coli tRNA Phe.

E. coli tRNAPhe has been labelled with fluorescein isothiocyanate taking advantage of the reactivity of this compound for primary aliphatic amino groups as exist in this tRNA as the modified base X(3-(3-amino-3-carboxypropyl)uracil). The extent of labelling was calculated as 1.6 nmole/A260 unit suggesting one dye molecule per tRNA. The FITC-tRNA showed full activity in aminoacylation and polypeptide synthesis. The absorption and fluorescence of the label respond markedly on addition of Mg++ to the tRNA. The label appears to be a sensitive probe of tRNAPhe tertiary structure.

Escherichia coli↗
PubMed1979

On the structure and conformational dynamics of yeast phenylalanine-accepting transfer ribonucleic acid in solution.

The solution structure of yeast tRNAPhe was investigated by using ethidium as a fluorescent probe in the D loop and the anticodon loop. For this purpose the dihydrouracils in position 16/17 and wybutine in position 37 were substituted by ethidium. The lifetimes and the time-dependent anisotropy of ethidium fluorescence were measured by pulsed nanosecond fluorometry. The kinetics of the transitions between different states of the tRNAPheEtd derivatives were determined by chemical relaxation measurements. It was found that the ethidium label irrespective of its position exhibits three different states called T1, T2 and T3 characterized by lifetimes tau 1 = 30 ns, tau 2 = 12 ns, and tau 3 = 3 ns. The lifetime differences are due to different accessibilities of ethidium for solvent quenching in the three states. Thus, there are three different defined structural environments of the ethidium in both the anticodon and the D loop. The distribution of the three states was measured as a function of Mg2+ concentration and temperature; it was found that state T3 is favored over states T2 and T1 by both increasing Mg2+ concentration and increasing temperature. The chemical relaxation kinetics exhibit a fast transition between T1 and T2 (10--100 ms) and a slow transition between T2 and T3 (100--1000 ms). The rates of both transitions depend likewise on Mg2+ concentration and temperature. The equilibrium and kinetic data clearly show the presence of strong and weak interactions between Mg2+ and tRNA. A cooperative model accounting for this behavior is developed. The ethidium probe behaves identically when located in different regions of the tRNA regarding both its distribution of states and its transition kinetics. This suggests that the different spectroscopic states report different conformations of the tRNA structure. The dependence of the three states on Mg2+ and spermine indicates that conformation T3 is closely related to or identical with the crystal structure. The rotational diffusion constants indicate that of all three states T3 is most extended while T2 is most compact. The thermodynamic analysis reveals that the strongly bound Mg2+ ions reduce both the activation entropy and enthalpy of all transitions. The weakly bound Mg2+ ions increase both the activation enthalpy and entropy of the slow transition between T2 and T3. It is suggested that the breaking of several intramolecular bonds, e.g., hydrogen bonds, is involved in this transition.

Ethidium↗
PubMed1976

On the interaction of seryl-tRNA synthetase with tRNA Ser. A contribution to the problem of synthetase-tRNA recognition.

By following the tryptophan fluorescence of yeast seryl-tRNA synthetase on addition of tRNA Ser it was observed that the number of binding sites for tRNA decreases from two to one with increasing temperature, ATP or KCl concentration. Concomitantly a considerable decrease of the apparent binding constant was observed. The variation in the number of binding sites is explained by the presence of at least one temperature and ionic strength sensitive binding site and one temperature and ionic strength independent binding site. Relaxation kinetic experiments revealed two binding processes: a fast one depending on tRNA concentration and ionic strength and a slow one, which appeared to be independent of tRNA concentration and ionic strength. Enzyme kinetic studies showed that the activity of seryl-tRNA synthetase strongly depends on the KCl concentration and exhibits a maximum at 0.2 M KCl. Based on the data from relaxation and enzyme kinetic experiments a model is suggested for the recognition process involving a first unspecific step where all tRNAs, cognate and non-cognate, are bound to the synthetase (scanning step). The identification of the cognate tRNA is then performed at the recognition site by a conformational transition of the tRNA . synthetase complex (identification step).

Amino Acyl-tRNA Synthetases↗
PubMed1976

Kinetic studies on the cleavage of adenovirus DNA by restriction endonuclease Eco RI.

The kinetics of cleavage of DNA from Adenovirus Type 1 (Ad1), Type 5 (Ad5) and Type 6 (Ad6) by restriction endonuclease EcoRI was investigated by quantitative evaluation of the fluorescence from ethidium stained DNA fragments separated on agarose gels. The apparent rate constants of cleavage at different cleavage sites have been determined and large differences in the cleavage rates of the individual sites within one type of DNA were found. From the kinetics of cleavage information on the sequence of the DNA fragments can be obtained. The order of the fragment A, B, C, D of Ad6 DNA obtained after complete cleavage by restriction endonuclease Eco RI was found to be A-D-C-B; the order of the corresponding fragments A, B, C of Ad1 and Ad5 DNA was found to be A-C-B.

Adenoviruses, Human↗
PubMed1975

Allosteric mechanism for codon-dependent tRNA selection on ribosomes.

We suggest that the interaction between a codon and its cognate tRNA induces conformational changes in the tRNA. We further suggest that sites on the ribosome preferentially bind tRNA in those conformations which require proper matching of codon and anticodon. According to this model, the codon functions as an allosteric effector which influences the conformation at various sites in the tRNA. This is made possible by the ribosome, which we suggest traps tRNA molecules in those conformation states that maximize the energy difference between cognate and noncognate codon-anticodon interactions. Studies of the interactions between tRNA molecules and their cognate codons in the absence of the ribosome have suggested that triplet-triplet interaction between codon and anticodon is far too weak to account for the specificity of the tRNA selection mechanism during protein synthesis. In contrast, we suggest that such affinity measurements do not adequately describe the interaction between a codon and its cognate tRNA. Thus, such experiments can not detect conformational changes in the tRNA, and, in particular, those stabilized by the ribosome.

Allosteric Regulation↗