Decreased imino proton exchange and base-pair opening in the IHF-DNA complex measured by NMR

Dhavan, Gauri; Lapham, Jon; Yang, Shu-Wei; Crothers, Donald M.

doi:10.1006/jmbi.1999.2690

Cited by 33 publications

(31 citation statements)

References 29 publications

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“…Thus, the opening of bubbles in the DNA duplex, when it is tightly wrapped around IHF, is much slower than the DNA bending times observed in our kinetics measurements, and also much slower than the opening͞closing times observed at a single base pair level for uncomplexed B-DNA (36)(37)(38).…”

Section: Discussioncontrasting

confidence: 50%

“…Recent determination of base pair lifetimes, monitored by NMR measurement of imino proton exchange kinetics (36,37), have shown that single A:T base pair opening rates in B-DNA bracket the measured DNA bending rate from 10°C to 30°C (Fig. 5c).…”

Section: Discussionmentioning

confidence: 75%

“…The opening͞closing dynamics of bubbles in a cognate DNA substrate bound to IHF, monitored with imino proton exchange measurements, yield concerted opening and closing times of Ϸ750 ms and Ϸ5 s, respectively, at 30°C, for bubbles of size up to Ϸ6 bp in the consensus region of the DNA sequence (36). Thus, the opening of bubbles in the DNA duplex, when it is tightly wrapped around IHF, is much slower than the DNA bending times observed in our kinetics measurements, and also much slower than the opening͞closing times observed at a single base pair level for uncomplexed B-DNA (36)(37)(38).…”

Section: Discussionmentioning

confidence: 99%

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Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

Kuznetsov¹,

Sugimura

Vivas³

et al. 2006

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

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Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNAbinding sites. Previous kinetics measurements on several DNAbending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending͞unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from Ϸ2 ms ؊1 at Ϸ37°C to Ϸ40 ms ؊1 at Ϸ10°C and correspond to an activation energy of Ϸ14 ؎ 3 kcal͞mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend͞kink DNA before forming a tight complex with IHF.DNA bending kinetics ͉ laser temperature jump ͉ protein-DNA interactions ͉ time-resolved FRET measurements

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Section: Discussioncontrasting

confidence: 50%

Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

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Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

Kuznetsov¹,

Sugimura

Vivas³

et al. 2006

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

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“…8; Dhavan et al 1999). Since the imino protons exchange only slowly from the closed base pair, these conditions are likely reporting on intrinsic exchange catalysis by the nitrogen of the accepting base pair, in a process involving one or several water molecules (Gueron et al 1987;Snoussi and Leroy 2001;Varnai et al 2004).…”

Section: C8mentioning

confidence: 99%

The global structures of a wild-type and poorly functional plant luteoviral mRNA pseudoknot are essentially identical

Cornish¹,

Stammler²,

Giedroc³

2006

RNA

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The helical junction region of a À1 frameshift stimulating hairpin-type mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) is characterized by a novel C27 Á (G7-C14) loop 2-stem 1 minor groove base triple, which is stacked on a C8+ Á (G12-C28) loop 1-stem 2 major groove base triple. Substitution of C27 with adenosine reduces frameshifting efficiency to a level just twofold above the slip-site alone. Here, we show that the global structure of the C27A ScYLV RNA is nearly indistinguishable from the wild-type counterpart, despite the fact that the helical junction region is altered and incorporates the anticipated isostructural A27 Á (G7-C14) minor groove base triple. This interaction mediates a 2.3-Å displacement of C8+ driven by an A27 N6-C8 + O2 hydrogen bond as part of an A (nÀ1) Á C + Á G-C n base quadruple. The helical junction regions of the C27A ScYLV and the beet western yellows virus (BWYV) pseudoknots are essentially superimposable, the latter of which contains an analogous A25 Á (G7-C14) minor groove base triple. These results reveal that the global ground-state structure is not strongly correlated with frameshift stimulation and point to a reduced thermodynamic stability and/or enhanced kinetic lability that derives from an altered helical junction architecture in the C27A ScYLV RNA as a significant determinant for setting frameshifting efficiencies in plant luteoviral mRNA pseudoknots.

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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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Decreased imino proton exchange and base-pair opening in the IHF-DNA complex measured by NMR

Cited by 33 publications

References 29 publications

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

The global structures of a wild-type and poorly functional plant luteoviral mRNA pseudoknot are essentially identical

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

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