Search PubMed⌕ Search

Biomedical subjects

D R Hare

Publications and source records attributed to D R Hare.

28 records · Page 2Linked to original sources
PubMed1984

Rotational dynamics of transfer ribonucleic acid: effect of ionic strength and concentration.

We have investigated the influence of ionic strength and nucleic acid concentration on the rotational Brownian motion of Escherichia coli tRNA1Val by studying the decay of the fluorescence polarization anisotropy (FPA) of intercalated ethidium on a nanosecond time scale. The rotational relaxation time tau R remains essentially constant as the ionic strength is varied from 2 to 100 mM at a tRNA concentration of 54 mg/mL. tau R also remains practically unchanged as the tRNA concentration is varied from 0.3 to 54 mg/mL at an ionic strength of 130 mM. Present hydrodynamic theories generally predict a more pronounced concentration dependence for rotational diffusion than we observe. This disagreement may result from a nonrandom distribution of the tRNA molecules in solution due to electrostatic interactions. By combining independent data from time-resolved nuclear Overhauser effect (NOE) cross-relaxation experiments and FPA experiments on the same tRNA, we are able to estimate the interproton spacing for the guanine N1-H and the uracil N3-H of the GU-50 base pair in E. coli tRNA1Val. This distance is 0.272 nm.

Escherichia coli↗
PubMed1984

Effect of Mg2+ on solution conformation of two different transfer ribonucleic acids.

We have investigated the effect of Mg2+ on the solution conformation of two different tRNAs by studying the decay of the fluorescence polarization anisotropy of intercalated ethidium on a nanosecond time scale. In the presence of endogenous Mg2+, yeast tRNAPhe and Escherichia coli tRNAVal1 exhibit similar behavior; i.e., the fluorescence from the intercalated ethidium decays biexponentially with lifetimes of approximately 25 and approximately 5 ns, and the fluorescence polarization anisotropy decays with a lifetime of approximately 25 ns. However, once Mg2+ is removed from the two tRNAs, their behavior is no longer similar. In the case of yeast tRNAPhe, it appears that titrating with Mg2+ restores the tRNA to the condition that it was in prior to the Mg2+ removal. This is not so for E. coli tRNAVal1, in which case titrating with Mg2+ results in a two-component anisotropy decay with lifetimes of approximately 25 and approximately 6 ns. Rudimentary calculations indicate that the 6-ns component does not result simply from a change in conformation of the tRNA. However, torsional motions in the tRNA facilitated by a torsion "joint" with a rigidity approximately 1/40 that of intact linear phi 29 DNA would yield a decay component on this time scale with about the right amplitude. We are thus left with the possibility that (after initially removing magnesium) titrating tRNAVal1 with Mg2+ leads to increased internal flexibility and a significant amplitude of a deformational relaxation mode. At any rate, there is no question that after removal of Mg2+ tRNAPhe and tRNAVal1 display quite different solution conformation behavior. These findings are in qualitative agreement with recent 500-MHz 1H NMR results from solutions of these two tRNAs.

Cations, Divalent↗
PubMed1984

Sequence-specific recognition of DNA: NMR studies of the imino protons of a synthetic RNA polymerase promoter.

We have synthesized both strands of a DNA duplex containing the consensus Pribnow promoter sequence TATAATG , flanked by GC base pairs to stabilize the ends of the helix. The stability of this duplex has been studied by using 1H nuclear magnetic resonance. The imino protons have been assigned by using the sequential nuclear Overhauser effect approach. Exchange rates have been monitored by using selective inversion recovery measurements. The helix is relatively unstable in the center of the AT-rich region even when surrounded by GC base pairs, and there is considerable asymmetry in the melting of the helix.

Base Sequence↗
PubMed1984

Sequence-specific recognition of DNA: assignment of nonexchangeable proton resonances in the consensus Pribnow promoter DNA sequence by two-dimensional NMR.

The resonances of most of the nonexchangeable protons of both + and - strands of the consensus Pribnow dodecamer d( CGTTATAATGCG ) have been assigned by two-dimensional nuclear magnetic resonance methods. Application of the two-dimensional nuclear Overhauser effect ( NOESY ) sequential connectivity method, combined with two-dimensional autocorrelated ( COSY ) spectra to reveal scalar-coupled protons, results in assignment of virtually all of the base and sugar protons, except the sugar C5 protons which are inadequately resolved. Analysis of the nuclear Overhauser data indicates that the helix assumes a fairly uniform B form conformation.

Base Sequence↗
PubMed1983

Assignment of the non-exchangeable proton resonances of d(C-G-C-G-A-A-T-T-C-G-C-G) using two-dimensional nuclear magnetic resonance methods.

A general method of assigning the non-exchangeable protons in the nuclear magnetic resonance spectra of small DNA molecules has been developed based upon two-dimensional autocorrelated (COSY) and nuclear Overhauser (NOESY) spectra in 2H2O solutions. Groups of protons in specific sugars or bases are identified by their scalar couplings (COSY), then connected spatially in a sequential fashion using the Overhauser effect (NOESY). The method appears to be generally applicable to moderate-sized DNA duplexes with structures close to B DNA. The self-complementary DNA sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been synthesized by the solid-phase phosphite triester technique and studied by this method. Analysis of the COSY spectrum and the NOESY spectrum leads to the unambiguous assignment of all protons in the molecule except the poorly resolved H5' and H5" resonances. The observed NOEs indicate qualitatively that, in solution, the d(C-G-C-G-A-A-T-T-C-G-C-G) helix is right-handed and close to the B DNA form with a structure similar to that determined by crystallography.

Base Sequence↗
PubMed1983

Sequence-specific recognition of deoxyribonucleic acid. Chemical synthesis and nuclear magnetic resonance assignment of the imino protons of lambda OR3 operator deoxyribonucleic acid.

Using solid-phase phosphite triester methods, we have synthesized both strands of the phage lambda OR3 DNA sequence, reannealed them, and studied the native operator duplex by high-resolution NMR at 500 MHz. At 7 degrees C the imino protons of the two terminal base pairs at each end have disappeared from the spectrum by exchange broadening. The 13 detectable imino resonances have been assigned to their respective base pairs in the duplex by using sequential nearest-neighbor NOE connectivity methods described previously. In cases where two imino protons overlap in the spectrum, spin diffusion was used to drive the cross-saturation further afield in order to produce second-order next-nearest-neighbor effects. The results show that the imino connectivity method can be used to unambiguously assign the imino proton spectrum of operator DNAs containing one to two full turns of the helix.

Bacteriophage lambda↗
PubMed1983

Nuclear magnetic resonance studies on structure and breathing dynamics of transfer RNA.

The hydrogen-bonded ring NH protons of the complementary base pairs in Escherichia coli isoleucine tRNA have been studied by 500 MHz nuclear magnetic resonance. These protons generate a set of about 30 well-resolved resonances in the -10p.p.m. to-15 p.p.m. region of the spectrum and can be observed in H2O solutions by Fourier transform NMR using the Redfield 214 observation pulse. Each of these low field protons has been assigned to its respective base pair in the tRNA by sequential nuclear Overhauser effect connectivity to its nearest neighbours. The helix-coil opening rates for several individual base pairs have been determined by monitoring the solvent-exchange contribution to the inversion recovery rate or the saturation recovery rate of the assigned ring NH peaks. Contrary to current theory, the two most rapidly opening base pairs in the molecule are located not at the helix termini but in the interior of helices.

Base Composition↗
PubMed1982

Nuclear Overhauser assignment of the imino protons of the acceptor helix and the ribothymidine helix in the nuclear magnetic resonance spectrum of Escherichia coli isoleucine transfer ribonucleic acid: evidence for costacked helices in solution.

In a previous study we showed that, in the low-field nuclear magnetic resonance (NMR) spectrum of Escherichia coli tRNAVal1, the hydrogen-bonded imino protons of the four Watson-Crick base pairs in the dihydrouridine helix could be assigned on the basis of their proximity to the imino proton of s4U8 by means of sequential Nuclear Overhauser (NOE) connectivity (Hare & Reid, 1982). In the present paper we have used the nearest-neighbor NOE technique to assign all the imino proton resonances of the acceptor helix and the ribothymidine helix of E. coli tRNAIle1. As reference points we used the GU-type base pairs located at positions 5 and 49 in this molecule which are readily identifiable in the NMR spectrum by virtue of containing two imino protons in the same base pair. From UG5 the imino protons of base pairs 4,3,2,1 and 6,7 could be assigned by through-space NOE connectivity. Similarly the imino protons of 50,51,52,53 were assigned by their spatial relationship to G psi 49. NOE connectivity also revealed a base pair stacked on the external side of GC53 which, by analogy with the crystal structure of yeast phenylalanine tRNA, is presumed to be the tertiary pair T54-A58. This was confirmed by NOE connectivity from the thymine methyl resonance. In addition to assigning 17 of the imino resonances in the low-field NMR spectrum of isoleucine tRNA, these NOE studies show that the acceptor helix and the ribothymidine helix are stacked on each other in solution in that base pairs 7 and 49 are directly connected in space.

Escherichia coli↗
PubMed1982

Direct assignment of the dihydrouridine-helix imino proton resonances in transfer ribonucleic acid nuclear magnetic resonance spectra by means of the nuclear Overhauser effect.

The NMR resonances from the hydrogen-bonded ring NH protons in the dihydrouridine stem of Escherichia colt tRNA1Val have been assigned by experiments involving the nuclear Overhauser effect (NOE) between adjacent base pairs. Irradiation of the 8-14 tertiary resonance produced a NOE to base pair 13. Irradiation of the CG13 ring NH produced NOEs to base pairs 12 and 14. Similarly, base pair 12 was shown to be dipolar coupled to 11 and 13, and base pair 11 was found to be coupled to 10 and 12. These sequential connectivities led to the assignment of CG13 at -13.05 ppm, UA12 at -13.84 ppm, CG11 at -12.23 ppm, and GC10 at -12.60 ppm. The results are compared with previous, less direct assignments for these four base pairs and with the expected proton positions from the crystal structure coordinates for this helix.

Escherichia coli↗