Global structure and mechanical properties of a 10-bp nucleosome positioning motif.

The method of DNA cyclization kinetics reveals special properties of the TATAAACGCC sequence motif found in DNA sequences that have high affinity for core histones. Replacement of 30 bp of generic DNA by three 10-bp repeats of the motif in small cyclization constructs increases cyclization rates by two orders of magnitude. We document a 13 degrees bend in the motif and characterize the direction of curvature. The bending force constant is smaller by nearly 2-fold and there is a 35% decrease in the twist modulus, relative to generic DNA. These features are the likely source of the high affinity for bending around core histones to form nucleosomes. Our results establish a protocol for determination of the ensemble-averaged global solution structure and mechanical properties of any approximately 10-bp DNA sequence element of interest, providing information complementary to that from NMR and crystallographic structural studies.

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

Integration Host Factor, IHF, is an E. coli DNA binding protein that imposes a substantial bend on DNA. Previous footprinting studies and bending assays have characterized several recognition sequences in the bacterial and lambda phage genome as unique in the way they are bound by IHF. We have chosen one of the lambda phage sites, H1, for study because it presents a small yet sequence-specific substrate for NMR analysis of the complex. A 19 base-pair duplex, H19, corresponding to the recognition sequence at the H1 site was constructed by isotopically labeling one of the strands with 15N. (1H, 15N) heteronuclear NMR experiments aided in assigning the imino proton resonances of the DNA alone and in complex with IHF. The NMR results are consistent with a mode of binding observed in the recent crystal structure of IHF bound to another of its sites from the lambda phage genome. Additionally, the dramatic change that IHF imposes on the imino proton chemical shifts is indicative of a severe deviation from canonical B-DNA structure. In order to understand the dynamic properties of the DNA in the complex with IHF, the exchange rates of the imino protons with the solvent have been measured for H19 with and without IHF bound. A drastic reduction in exchange is observed for the imino protons in the IHF bound DNA. In the DNA-protein complex, groups of adjacent base-pair exchange at the same rate, and appear to close more slowly than the rate of imino proton exchange with bulk water, since their exchange rate is independent of catalyst concentration. We infer that segments of the double helix as large as 6 bp open in a cooperative process, and remain open much longer than is typical for opening fluctuations in naked duplex DNA. We discuss these results in terms of the specific protein-DNA contacts observed in the crystal structure.

Measurement of diffusion constants for nucleic acids by NMR.

Pulsed field-gradient NMR experiments can be used to measure the diffusion constants of nucleic acids. The diffusion constants measured in this way for double-helical DNAs of defined length agree well both with theory and with measurements done using other techniques. When applied to RNAs, this experiment easily distinguishes duplex RNAs from RNA hairpins and thus it can solve one of the perennial problems faced by RNA spectroscopists, i.e. assessing whether their samples are monomeric or not.

RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation.

Large quantities of RNA for study by NMR and X-ray crystallography can be produced by transcription reactions in vitro using T7 bacteriophage RNA polymerase. A limitation on producing RNA with this polymerase has been the strong dependence of the yield of the transcription reaction on the sequence at the 5' end of the RNA produced. We report a procedure for obtaining large quantities of enzymatically synthesized RNA from T7 RNA polymerase that has no dependence on the 5' end sequence of the target RNA. Ribonuclease H has been shown previously (Inoue H, Hayase Y, Iwai S, Ohtsuka E, 1987, FEBS Lett 215:327-330) to cleave RNA site specifically using 2'-O-methyl RNA/DNA chimeras to direct the cleavage site. We show that 2'-O-methyl RNA nucleotides on the 5'-side of the DNA nucleotides in the chimera are not essential for site-specific cleavage. This allowed us to design the method such that the same 2'-O-methyl chimera may be used to process any RNA sequence. We have adapted this reaction to the cleavage of NMR-scale quantities of RNA at high yield. RNA is synthesized using T7 RNA polymerase with a 15-nt high-yielding leader sequence at the 5' end, and then this sequence is cleaved off with the RNase H cleavage reaction. The cleaved RNA has 3'-hydroxyl and 5'-phosphate ends, so that the products can be used directly as substrates for ligation by T4 DNA ligase. We show that the cleavage reaction occurs efficiently in solution and on a solid streptavidin/agarose matrix. We report an example in which we are able to improve transcription yield by more than five-fold using this technique in the synthesis of a 15N isotopically labeled hairpin found in the Crithidia fasciculata spliced leader RNA. We are able to obtain a 0.5-mM NMR sample from this inherently poorly transcribing sequence, while minimizing the amount of isotopically labeled rNTPs used to produce it. The NMR spectroscopic results are consistent with the predicted RNA secondary structure.

Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C- and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and 15N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional [15N] 1H heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 microM concentration range, with a total of 30 nmol or approximately 40 micrograms of RNA in approximately 150 microliters, give strong NMR signals in a short accumulation time. The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems. This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure.