Study of transfer ribonucleic acid unfolding by dynamic nuclear magnetic resonance

Johnston, Paul D.; Redfield, Alfred G.

doi:10.1021/bi00517a008

Cited by 64 publications

(54 citation statements)

References 26 publications

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“…The imino protons can recover their magnetization after saturation by spin-lattice relaxation (magnetic contribution) and by exchange with unperturbed solvent water (chemical contribution). Previous studies on transfer RNA (11,23,24) and DNA fragments (13)(14)(15)(16)(17) have demonstrated that the magnetic contribution predominates below room temperature and has a small activation energy, whereas the chemical contribution predominates above room temperature and has a large activation energy. We have determined the temperature dependence of the saturation recovery lifetimes of the AATT 12-mer duplex and the TATA 12-mer duplex between 0C and 60'C, and these are tabulated in Table 1.…”

Section: Bmentioning

confidence: 98%

Sequence dependence of hydrogen exchange kinetics in DNA duplexes at the individual base pair level in solution.

Patel¹,

Ikuta²,

Kozlowski³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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The kinetics for hydrogen exchange at individual base pairs in self-complementary deoxydodecanucleotide duplexes have been estimated from NMR saturation recovery measurements on the resolved imino protons as a function of temperature.The imino protons of dA-dT base pairs in the center of the fully alternating d(C-G-C-G-T-A-T-A-C-G-C-G) duplex exchange a factor of 2-to 3-fold faster than the corresponding protons at the same positions in the partially alternating d(C-G-C-G-A-A-T-T-C-G-C-G) duplex. These exchange parameters are a direct measure of the rate constants for transient opening of individual dA-dT base pairs in the dodecanucleotide duplexes and demonstrate faster opening kinetics for the "TATA" box region compared to the related "AATT" segment.Recent advances in oligonucleotide synthesis have led to the preparation of milligram quantities of DNA fragments of precise sequence containing at least one turn of helix (1). One such example is the self-Scheme I which has been extensively analyzed by single-crystal x-ray analysis in the solid state (2) and high-resolution NMR spectroscopy in aqueous solution (3). Both techniques have established that the central A-T-rich tetranucleotide segment shows conformational polymorphism in the duplex state as a function of temperature and solvent (2, 3).These observations initiated the present experiments, which probe the effect of sequence on the conformation and dynamics of DNA duplexes in solution. The completely alternating self-was synthesized by solid-state methods, and the effect of sequence variations in the center of the duplex was probed by comparative studies of the AATT 12-mer and TATA 12-mer duplexes.Redfield et al. have introduced nuclear Overhauser effect (NOE) and saturation recovery methods to probe the conformation and dynamics of transfer RNA in solution (4). They have demonstrated intra-and inter-base-pair NOEs involving exchangeable and nonexchangeable protons and have correlated the magnitude of the NOE effect with the inverse sixth power dependence of the interproton distance between the saturated and observed resonances. This distance-measuring methodology has wide applications, including its extension to elucidate conformational features of DNA fragments (3), synthetic DNAs (5), and antibiotic-DNA complexes (6) in solution.Hydrogen-deuterium exchange stop-flow measurements have demonstrated that the exchange kinetics of the Watson-Crick imino protons are sensitive markers of the dynamics of base pair opening in the interior of nucleic acid duplexes (7). NMR also can monitor hydrogen exchange, and the early research focused on line width changes of the exchangeable imino protons in the continuous wave mode to evaluate the kinetics of base pair-opening reactions (8, 9). The introduction of Fourier transform methods with H20 solvent suppression (10) readily permitted the incorporation of variable time delays between the saturation and observation pulses to monitor the kinetics of magnetization recovery. The initial measurements were made on...

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Section: Bmentioning

confidence: 98%

Sequence dependence of hydrogen exchange kinetics in DNA duplexes at the individual base pair level in solution.

Patel¹,

Ikuta²,

Kozlowski³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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“…Inspection of the 1 H NMR spectra of the bacteriophage T2 and the frameshifting pseudoknots suggests that there are significant differences in the rates at which the imino protons within the base pairs exchange with solvent (Fig+ 4), particularly for those base pairs located at the junction of the pseudoknot stems+ The relationship between solvent exchange rate and dynamic features of RNA such as the opening frequency of base pairs can be quite complex+ When the base pair dissociation constant is much less than one, and the rate of imino proton exchange is significantly greater than the rate of base pair closing, the imino proton exchanges with solvent each time the base pair opens (referred to as an "EX1" mechanism)+ Alternatively, a base pair may open many times before proton exchange occurs; in this case the rate-limiting step is the base-catalyzed exchange with solvent+ The former mechanism has been observed in studies of tRNA (Hurd & Reid, 1980;Johnston & Redfield, 1981;Choi & Redfield, 1995) and a protein-DNA complex (Dhavan et al+, 1999), whereas the latter has been observed in several DNAs and drug-DNA complexes, in which case, the rate of base pair opening may be one to two orders of magnitude higher than the imino proton exchange frequency (reviewed by Guéron & Leroy, 1995)+ The observation of relatively rapid imino proton exchange for a particular base pair within an RNA structure therefore implies that either the base pair has a relatively rapid opening frequency or the efficiency of exchange is accelerated by the presence of a catalyst+ Imino proton exchange rates for the bacteriophage T2 pseudoknot and the representative frameshifting pseudoknots were estimated using a saturation transfer method+ For the bacteriophage T2 pseudoknot, imino proton exchange rates for the stem base pairs are all relatively slow, in the range of 0+8 to 4+2 Hz (Fig+ 8; ), and estimated imino proton exchange rates (K HX ) for guanosine N1 and uridine N3 imino groups within base-paired nucleotides+ Theta is the angle between the imino N-H bond vector and the major axis of inertia of the pseudoknot+ The imino resonances of G4 and G10 and G5 and G13 overlap in both the 1 H and 15 N dimensions; reported relaxation rates for these nuclei are for the composite (overlapping) peaks+ Table 3)+ Interestingly, the exchange rates of imino protons at the junction of the stems are not much different from those in the central regions of the stems+ In the SRV frameshifting pseudoknot (Fig+ 8; Table 4), exchange rates for most of the base-paired imino protons are in the range of 1 to 2 Hz, with the exceptions of G16 at the junction of the stems (;50 Hz), G3 at the end of stem 1 (;9 Hz), and U34 at the junction of the two stems (;300 Hz)+ In the VPK frameshifting pseudoknot and the hybrid frameshifting pseudoknot, the imino protons of the base pairs adjacent to the junction of the stems are unobserved, indicating solvent exchange rates of greater than 300 Hz+ The data therefore suggest that the opening frequency of the base pairs at the junction of the stems in the bacteriophage T2 pseudoknot is significantly slower than the corresponding base pairs in the frameshifting pseudoknots+…”

Section: Comparison Of Imino Proton Exchange Ratesmentioning

confidence: 99%

Comparative studies of frameshifting and nonframeshifting RNA pseudoknots: A mutational and NMR investigation of pseudoknots derived from the bacteriophage T2 gene 32 mRNA and the retroviral gag-pro frameshift site

Wang

Wills

et al. 2002

RNA

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Mutational and NMR methods were used to investigate features of sequence, structure, and dynamics that are associated with the ability of a pseudoknot to stimulate a -1 frameshift. In vitro frameshift assays were performed on retroviral gag-pro frameshift-stimulating pseudoknots and their derivatives, a pseudoknot from the gene 32 mRNA of bacteriophage T2 that is not naturally associated with frameshifting, and hybrids of these pseudoknots. Results show that the gag-pro pseudoknot from human endogenous retrovirus-K10 (HERV) stimulates a -1 frameshift with an efficiency similar to that of the closely related retrovirus MMTV. The bacteriophage T2 mRNA pseudoknot was found to be a poor stimulator of frameshifting, supporting a hypothesis that the retroviral pseudoknots have distinctive properties that make them efficient frameshift stimulators. A hybrid, designed by combining features of the bacteriophage and retroviral pseudoknots, was found to stimulate frameshifting while retaining significant structural similarity to the nonframeshifting bacteriophage pseudoknot. Mutational analyses of the retroviral and hybrid pseudoknots were used to evaluate the effects of an unpaired (wedged) adenosine at the junction of the pseudoknot stems, changing the base pairs near the junction of the two stems, and changing the identity of the loop 2 nucleotide nearest the junction of the stems. Pseudoknots both with and without the wedged adenosine can stimulate frameshifting, though the identities of the nucleotides near the stem1/stem2 junction do influence efficiency. NMR data showed that the bacteriophage and hybrid pseudoknots are similar in their local structure at the junction of the stems, indicating that pseudoknots that are similar in this structural feature can differ radically in their ability to stimulate frameshifting. NMR methods were used to compare the internal motions of the bacteriophage T2 pseudoknot and representative frameshifting pseudoknots. The stems of the investigated pseudoknots are similarly well ordered on the time scales to which nitrogen-15 relaxation data are sensitive; however, solvent exchange rates for protons at the junction of the two stems of the nonframeshifting bacteriophage pseudoknot are significantly slower than the analogous protons in the representative frameshifting pseudoknots.

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“…This appears to be a characteristic of all FUra-substituted tRNAs, and may indicate that peak A corresponds to an invariant base substituted by fluorouracil. Redfield and collaborators (Johnson and Redfield, 1981;Tropp and Redfield, 1981) have observed a resonance in the NMR spectra of several tRNAs that also exhibits a hypersensitivity to changes in Mg**. This peak has been assigned to the imino proton at N(l) of Y55.…”

Section: Ion Binding ^ Fura-substituted Trnamentioning

confidence: 99%

“…The thermal unfolding sequence of tRNA^he has recently been studied using imino proton exchange (Johnson and Redfield, 1981;Roy and Redfield, 1983 Reid (1981) where the anticodon helix melts before unfolding of tertiary structure occurs.…”

Section: Meltingmentioning

confidence: 99%

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19F NMR studies of 5-fluorouracil-substituted Escherichia coli transfer RNAs: solution structure and codon-anticodon interaction

Gollnick

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Study of transfer ribonucleic acid unfolding by dynamic nuclear magnetic resonance

Cited by 64 publications

References 26 publications

Sequence dependence of hydrogen exchange kinetics in DNA duplexes at the individual base pair level in solution.

Sequence dependence of hydrogen exchange kinetics in DNA duplexes at the individual base pair level in solution.

Comparative studies of frameshifting and nonframeshifting RNA pseudoknots: A mutational and NMR investigation of pseudoknots derived from the bacteriophage T2 gene 32 mRNA and the retroviral gag-pro frameshift site

19F NMR studies of 5-fluorouracil-substituted Escherichia coli transfer RNAs: solution structure and codon-anticodon interaction

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