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Biomedical subjects

C Urbanke

Publications and source records attributed to C Urbanke.

63 records · Page 4Linked to original sources
PubMed1981

The effect of several nucleic acid binding drugs on the cleavage of d(GGAATTCC) and pBR 322 by the Eco RI restriction endonuclease.

The endonucleolytic action of the EcoRI restriction enzyme on the double-stranded oligonucleotide d(GGAATTCC) and the supercoiled plasmid DNA pBR 233 is inhibited by actinomycin D, ethidium bromide, proflavin, distamycin A and netropsin. Half-maximal inhibition is observed at around 100 microM concentrations for the intercalating drugs, and around 0.1 to 1 microM concentrations for netropsin and distamycin A. The inhibitory activity of these drugs can be correlated with their affinity to the oligonucleotide and the plasmid DNA. Since at high concentrations of the drugs a complete inhibition is observed, it is concluded that the effect of the drugs on the stereochemistry of the EcoRI site is such that recognition is excluded.

Circular Dichroism↗
PubMed1981

Transient cleavage kinetics of the Eco RI restriction endonuclease measured in a pulsed quench-flow apparatus: enzyme concentration-dependent activity change.

We report measurements of the cleavage rate of pBR 322 plasmid DNA by the restriction endonuclease Eco RI as a function of enzyme and DNA concentration. The reaction, which at high excess of enzyme over DNA occurs between 0.2 and 5 seconds, was studied by the means of a microprocessor controlled pulsed quench-flow apparatus. Enzyme concentrations were between 1 and 100 nM with DNA concentrations being 3 to 6 nM (specific Eco RI sites). The catalytic constants for cleavage of the first and second phosphodiester bonds as measured at high enzyme concentration both have the same value of 0.35 sec-1 and 21 degrees C. At enzyme concentrations comparable to or less than DNA concentration, the rate of the first cleavage is proportional to enzyme concentration, while the second step is independent of concentration. At approx. 10 nM Eco RI endonuclease concentration, a rate increase shows up in both the first and the second cleavage. We suggest that this increase is due to the tetramerization reported by Modrich & Zabel1, which occurs in this concentration range.

DNA Restriction Enzymes↗
PubMed1980

Aminoacyl transfer ribonucleic acid binding site of the bacterial elongation factor Tu.

Hydrolysis protection experiments were used for a quantitative determination of the binding of several aminoacyl-tRNAs to the Escherichia coli elongation factor Tu. The observed differences could not be rationalized in terms of structural properties of the tRNAs. The experimental results support, however, a model according to which the differences in the affinity of naturally occurring aminoacyl-tRNAs are determined mainly by the nature of the amino acid esterified to the tRNA. Aminoacyl-tRNAs with polar amino acid side chains are bound less strongly than those with apolar ones. This model is substantiated by results obtained with misacylated and modified aminoacyl-tRNAs. Furthermore, it could be shown that the aminoacyl group of the aminoacyl-tRNA must be in the L configuration; EF-Tu in this way prevents blocking of the ribosomal A site or even incorporation of D-amino acids into protein. The data have been used for a schematic description of the structure of a part of the aminoacyl-tRNA binding site of the bacterial elongation factor Tu.

Carbon Radioisotopes↗
PubMed1978

A novel conformational change of the anticodon region of tRNAPhe (yeast).

The temperature dependence of the fluorescence of the Y-base of tRNAPhe (yeast) was investigated kinetically by the temperature jump method. In the range between -15 degrees C and +30 degrees C A NOVEL CONFORMATIONAL TRANSITION OF THE TRNA could be characterized. This conformational change was found in the absence of any artificial label; it is a characteristic property of tRNAPhe in its native structure. This transition accounts for 30% of the total fluorescence change. Its activation enthalpy is 16 kcal/mole (67 kJ/mole), and the transition enthalpy is between -2 kcal/mole and +2 kcal/mole (+/-8 kJ/mole). A model is represented in which this transition can be explained by a a change in the stacking pattern of the anticodon loop. The experimental findings are discussed with respect to several hypotheses about the molecular mechanism of protein biosynthesis which postulate conformational rearrangements of the anticodon loop.

Anticodon↗
PubMed1978

The binding of kirromycin to elongation factor Tu. Structural alterations are responsible for the inhibitory action.

The influence of kirromycin on the elongation factor Tu (EF-Tu) in its binary and ternary complexes was investigated. The equilibrium constant for the binding of the antibiotic to EF-Tu . GDP and EF-Tu . GTP was determined by circular dichroism titrations to be 4 x 10(6) M-1, and to EF-Tu . GTP . aa-tRNA by a combination of circular dichroism titrations and hydrolysis protection experiments to be 2 x 10(6) M-1. In the presence of kirromycin the binding of aminoacyl-tRNAs to EF-Tu . GTP is weakened by a factor of two. The antibiotic changes the conformation of the ternary complex in such a way that the aminoacyl moiety of the aminoacyl-tRNA is more accessible to the non-enzymatic hydrolysis. It is concluded that this structural alteration is responsible for the inhibitory action of the antibiotic.

Anti-Bacterial Agents↗
PubMed1977

Ternary complex formation between elongation factor Tu, GTP and aminoacyl-tRNA: an equilibrium study.

The equilibria between the elongation factor Tu-GTP complex (EF-Tu-GTP) from Escherichia coli and tyrosyl-tRNATyr from E. coli as well as phenylalanyl-tRNAPhe and seryl-tRNASer from yeast were studied using a novel procedure, which takes advantage of the protective effect of ternary complex formation on the stability of theaminoacyl bond against non-enzymatic hydrolysis. At 25 degrees C and at pH 7.4 tyrosyl-tRNATyr, phenylalanyl-tRNAPhe and seryl-tRNASer are bound with binding constants of 0.7 X 10(7) M-1, 5.0 X 10(7) M-1 and 0.5 X 10(7) M-1 respectively. The binding of aminoacyl-tRNA to EF-Tu-GTP has a negative deltaH of the order of 10 kcal/mol (42 kJ/mol). Complex formation is dependent on ionic strength: with 0.1 M KCl Kass = 0.8 X 10(7) M-1, with 0.5 M KCl Kass = 0.2 X 10(7) M-1 was determined for the binding of Tyr-tRNATyr.

Calorimetry↗
PubMed1975

Tertiary structure of tRNAPhe (yeast): kinetics and electrostatic repulsion.

Conditions were established that allowed the observation of the unfolding of the tertiary structure of tRNAPhe (yeast) without the interference of either secondary structure or low salt aberrant structures. Relaxation kinetics of tertiary structure melting show that the reaction proceeds according to co-operative all-or-none mechanism. The negative activation enthalpy of formation (delta H+ + R = -14 +/- 5 kcal/mol, -59 +/- 21 kJ/mol) implies a fast pre-equilibrium preceding the rate-limiting step. The ionic strength dependence of the corresponding rate constant demonstrates that most of the electrostatic repulsion characteristic of tertiary structure folding is overcome before the rate-limiting step is reached. On the other hand, most of the stabilizing enthalpy change occurs after the rate-limiting step. At the usual ionic strength (0.1 M Na+) tertiary structure folding is about 100 times slower than double-helix formation. Extrapolation of the rate constants to high ionic strengths, however, indicates that the dynamic differences between secondary and tertiary structure are only due to electrostatic repulsion. The stabilization of tertiary structure by alkaline salts is increased by decreasing the cationic radius. Double helices show virtually no dependence on the radius of monovalent cations. This indicates considerable geometric restrictions for the stabilization of tertiary structure.

Kinetics↗