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Biomedical subjects

D R Kearns

Publications and source records attributed to D R Kearns.

At least 91 records · Page 5Linked to original sources
PubMed1977

Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids.

The mechanism of the enhancement of the fluorescence of ethidium bromide on binding to double helical RNA and DNA has been investigated. From an examination of the effect of different solvents on the fluorescence lifetime, quenching of fluorescence by proton acceptors, and the substantial lengthening of lifetime observed upon deuteration of the amino protons, regardless of the medium, we conclude that proton transfer from the excited singlet state is the process primarily responsible for the approximately equal to 3.5-fold increase in the lifetime of free ethidium bromide in going from H2O to D2O; the fact that addition of small amounts of water to nonaqueous solvents decreases the fluorescence whereas addition of small amounts of D2O enhances the fluorescence; and the enhancement of the ethidium bromide triplet state yield on binding to DNA. Other proposed mechanisms are shown to be inconsistent with our findings.

Chemical Phenomena↗
PubMed1977

Effect of magnesium and polyamines on the structure of yeast tRNAPhe.

The effect of magnesium and polyamines (spermine, spermidine, putrescine and cadaverine) on the structure of yeast tRNAPhe has been investigated. It is found that magnesium induces structural changes and stabilizes hydrogen bonds in the temperature range 22--44 degrees C in 0.17 M sodium. The number of Mg2+ which affect tRNA structure increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C and the number of additional base pairs formed in the presence of magnesium increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C. The spectral changes are more-or-less sequential. The polyamine spermine stabilizes some, but not all, of the structural features stabilized by magnesium at 44 degrees C, and the combination of magnesium and spermine, at low levels, is more effective than either cation alone in stabilizing tRNA structure. Comparison of the effects of spermine, spermidine, putrescine and cadaverine indicates that it is the asymmetric triamine unit which is important in the stabilization. Some spectral changes induced by magnesium can be assigned to stabilization of specific tertiary structure interactions and to alteration of stacking adjacent to U8-A14.

Magnesium↗
PubMed1977

Manganese(II) as a paramagnetic probe of the tertiary structure of transfer RNA.

The effect of manganese on both the low field (10--15 ppm) and the high field (o--3 ppm) NMR spectra of unfractionated tRNA and yeast tRNAPhe has been investigated. Trace amounts of Mn2+ cause selective broadening of resonances which are assigned to specific tertiary interactions. The order in which resonances broaden is the same as the order in which they are stabilized by the addition of magnesium, namely s4U8 - A14, U33 and A58 - T54. From this we conclude that three of the strong binding sites probably are the same for both Mn2+ and Mg2+, and that these sites are located close to the tertiary interactions which are stabilized by the strongly bound metals. The broadening data, taken in conjunction with published X-ray data on yeast tRNAPhe, permit us to suggest some plausible locations for the strong binding sites.

Binding Sites↗
PubMed1977

High resolution proton nuclear magnetic resonance investigation of the structural and dynamic properties of d(C15A15)-d(T15G15).

The high resolution proton nuclear magnetic resonance (NMR) spectra of the synthetic DNA block polymer d(C15A15)-d(T15G15) were studied in order to more completely understand telestability in DNA, and to provide fundamental NMR data on DNA helices and random coils. Spectra were measured in the spectral region from 0 to 15 ppm downfield from the usual standard, sodium 4,4-dimethyl-4-silapentane-1-sulfonate(DSS), at various temperatures (24-98 degrees C) in solution containing either moderate or high ionic strength. The effect of actinomycin binding to the block polymer also was studied. The major conclusions derived from this study are as follows: (1) The majority of base pairs in the AT helix of the block polymer have the same conformation as in d(A)n-d(T)25 and d(A)21-d(T)21. (2) The conformation of the GC helix in the block polymer is different from the AT helix and this perturbs the conformation of three or four A-T base pairs at the junction of the AT-GC helix. (3) The conformation of the AT helix is unaffected by salt over the range examined (approximately 0.04 - approximately 2 M), but the conformation of the GC helix changes. (4) There are subtle changes in the conformation of the AT helix as the temperature is increased and resonances characteristic of the random coil and the double-helical state can be simultaneously observed. (5) Binding of actinomycin, which is specific for the GC helix, induces quite large (over 1 ppm) upfield shifts of the resonances from the GC base pairs. This is consistent with an intercalation model in which actinomycin D (Am) is assymetrically sandwiched between two GC base pairs in such a manner that overlap with the guanosine residues is greater than with the neighboring cytidines. (6) The presence of the drug may also perturb A-T base pairs located near the AT-GC junction, but it has no effect on the majority of the AT pairs. However, as expected, Am elevated the Tm of the AT helix, even though it binds to the other end of the DNA.

DNA↗
PubMed1976

Quantitative determination of the number of secondary and tertiary structure base pairs in transfer RNA in solution.

Resonances in the low-field (11-15 ppm) nuclear magnetic resonance spectrum (NMR) of tRNA molecules arise from secondary and tertiary structure base pairs (1 resonance for each base pair) as well as tertiary structure hydrogen bonds. An accurate method for integrating the low-field spectra has been developed and applied to seven different tRNA. In the presence of high levels of magnesium (10 mM free magnesium) the number of resonances (base pairs) per molecule is typically 3-4 more than the number predicted by the cloverleaf model. These results confirm our recent proposal that, under proper conditions, most tRNA exhibit 3-4 tertiary structure interactions in solution, which are also observed in x-ray diffraction studies of yeast tRNAPhe. In addition to common resonances in the 11-15 ppm region, there are common resonances at 10.5 and 9.5 ppm. A critique of methods used to integrate the low-field spectra is given and possible sources of error are indicated. The discrepancy between our present results and previous studies, which indicated that the number of base pairs per molecule was close to the number predicted by the cloverleaf model, can be attributed partly to differences in magnesium concentration and partly to inaccuracies inherent in the integration methods used.

Escherichia coli↗
PubMed1976

Nuclear magnetic resonance investigation of the base-pairing structure of Escherichia coli tRNATyr monomer and dimer conformations.

The structures of the Escherichia coli tyrosine tRNA monomer and dimer have been investigated by high-resolution nuclear magnetic resonance (NMR). At 23 degrees C the monomer contains 26 +/- 2 base pairs and the low-field NMR spectrum (11.7-15 ppm) can be accounted for in terms of the cloverleaf structure (23 base pairs) and three additional resonances that are assigned to tertiary structure base pairs. Assignments suggested for the various resonances are consistent with thermal denaturation studies in low-salt solutions. Under these conditions the temperature dependence of the spectrum can be interpreted in terms of sequential unfolding of the cloverleaf structure with the minor and dihydrouridine stems melting first, followed by the T psi C stem, the anticodon stem, and finally the amino acceptor stem. Certain features of the tertiary structure of tRNATyr are similar to other tRNA, but some details of the folding must be different, since no resonance from the S4U8-A14 tertiary base pair is observed. The tRNATyr dimer contains only 20 +/- 2 base pairs per tRNA (40/dimer) at 23 degrees C and a good account of the low-field NMR spectrum can be given in terms of a secondary structure in which bases of the T psi C stem and loop are involved in inter-molecular base pairing. Formation of the dimer requires opening of the hU and T psi C stems, but not the anticodon or amino acid acceptor stems, and this fits well with relative stabilities observed for these stems in the monomer. The model also provides an explanation for the formation of 2n-mers, that were stable enough to be separated by gel electrophoresis at room temperature (10 mM Mg2+). Experimental conditions required for interconversion of monomer and dimer are also described.

Base Sequence↗
PubMed1975

Specific conversion of s4 U to U in Escherichia coli tRNA by iodate oxidation.

The minor nucleoside 4-thiouridine in Escherichia coli tRNA is transformed selectively to uridine by iodate oxidation at acidic pH. The four major nucleotides were found to be inert under these conditions. The iodate oxidation appears to be more specific than the previous conversion methods reported, and has the advantage that it does not affect the chargeability of most tRNA.

Escherichia coli↗
PubMed1975

Identification of a unique ethidium bromide binding site on yeast tRNAPhe by high resolution (300 MHz) nuclear magnetic resonance.

The binding of ethidium bromide to yeast tRNAPhe has been studied by high resolution (300 MHz) proton nuclear magnetic resonance. Under appropriate experimental conditions one ethidium bromide is bound to each tRNA and two resonances from ring NH protons are shifted upfield. These observations taken in conjunction with the low-field spectrum of yeast tRNAPhe show that the unique ethidium bromide binding site is located between base pairs AU6 and AU7 of the amino acid acceptor stem. This information should be of value in understanding the way in which ethidium bromide binding alters the biochemical properties of the tRNA molecules.

Binding Sites↗