Nucleolin promotes secondary structure in ribosomal RNA (original) (raw)
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The Journal of Physical Chemistry B, 2004
Infrared (IR) and vibrational circular dichroism (VCD) spectra of single-stranded poly(rA) and poly(rU) RNA polynucleotides and double-and triple-stranded RNA helices were recorded and interpreted on the basis of density functional theory (DFT) at the BPW91/6-31G** level. Ab initio computations were performed for smaller fragments and extended to longer oligomers via transfer of atomic property tensors. The normal mode partial optimization method developed lately was found convenient for relaxation of the fragment geometries. Most of observed spectral features could be assigned to vibrations of the adenine, uracil, and sugar-phosphate chromophores; in particular, the VCD band shapes could be explained on the basis of specific spatial interactions. Thus, the VCD technique proved to be sensitive to various RNA conformational types (random coil, single strand, duplex, and triplex) in solution, spectra of which could be reliably modeled. The effect of the solvent could be only partially included, and ambiguity remains in the model structure used for the triplex RNA form.
Characterization of Tertiary Folding of RNA by Circular Dichroism and Urea
Current Protocols in Nucleic Acid Chemistry, 2001
Since X-ray crystallography of tRNA was first performed in the 1970s, the study of RNA folding has extended into the characterization of tertiary organization. Although circular dichroism (CD) spectroscopy cannot yet provide structural detail about tertiary organization, it can be used to monitor RNA tertiary folding transitions, which may not be observable by absorbance spectroscopy (Pan and Sosnick, 1997). With the use of computer-controlled titrators, data can be acquired rapidly and accurate thermodynamic parameters can be obtained over a wide variety of conditions. Hence, CD provides a nice complement to site-resolved methods such as complementary oligonucleotide hybridization (Zarrinkar and Williamson, 1994), hydroxyl radical footprinting (Sclavi et al., 1997), or chemical modification (Banerjee and Turner, 1995). This unit provides a basic outline for using CD and urea to measure and characterize tertiary RNA folding transitions to extract thermodynamic parameters. BASIC PROTOCOL 1 MEASUREMENT OF A CIRCULAR DICHROISM (CD) SPECTRUM The CD spectrum provides information about the amount of secondary and tertiary structure in the RNA. The changes in the spectrum can be used to identify folding transitions and determine their thermodynamic parameters. Some of these transitions, in particular tertiary transitions, may be unobservable by more standard absorbance measurements. Materials Unfolded RNA sample UV-VIS spectrophotometer 1-cm-path-length quartz cuvette 90°C water bath Circular dichroism (CD) spectrometer capable of far-UV measurements (e.g., Jasco or AVIV Associates) Magnetic stir bar to fit cuvette Additional reagents and equipment for RNA renaturation (UNIT 6.3) Prepare sample 1. Prepare an unfolded RNA sample with absorbance at 260 nm (A 260) of ∼0.3 to 0.8 AU for optimal signal-to-noise ratio. This corresponds to ∼10 to 20 µg/mL when using a 1-cm-path-length cuvette (1.5 mL). Increase concentration accordingly for shorter path lengths. The same cuvette can be used for the UV-VIS and CD spectrophotometers. Decreasing the RNA concentration will extend measurements further into the UV, but this also decreases the signal. Increasing the concentration will increase the signal, but this decreases the amount of light passing through the sample, and hence lowers the signal quality. Keep buffer and chloride concentrations to a minimum as these reagents absorb in the far-UV regions. 2. Perform a renaturing step (UNIT 6.3) involving heating to 90°C for 3 min, to remove residual structure formed during purification steps.
1982
The helix content of rRNA species (Escherichiu coli, Calduriellu ucidophila, rat liver) and the G . C content of their bihelical domains have been investigated by chemical modification of uracil and cytosine residues with probes specific for sterically exposed bases. By using radioactively labelled rRNA, G . C base pairs and the sum of A . U plus G + U base pairs have been quantified assuming that they are numerically identical with the unreactive cytosine and uracil rings, respectively. Exposed uracil bases were probed by their conversion to alkali-labile. nonultraviolet-absorbing sulphonated adducts, with 1.33 M bisulfite pH 7, at 20 "C; the adducts can be separated from unreacted uracil, and quantified, by cation-exchange chromatography of RNase T2 plus pancreatic RNase digests of bisulfite-modified rRNA. Exposed cytosines were probed by their conversion to methoxyaminated, alkalistable, derivatives with 1 M methoxyamine. pH 5.5. at 37'C, and quantified by monitoring the CMP/AMP radioactivity ratio after alkaline hydrolysis of modified rRNA. Exposed uracil rings can also be estimated spectrophotometrically by the alkali-catalyzed reversal of the non-ultraviolet-absorbing sulphonated adducts after separation of the latter from unreacted uracil. The cytosine deamination reaction, catalyzed by bisulfite at pH 6, has also been investigated and found to exhibit little specificity for sterically exposed bases of rRNA, the (G + C)richer rRNA species of C. ucidophila being considerably less susceptible to non-specific deamination than the (G + C)-poorer rRNA of E. coli. A high degree of congruence is shown to exist between results obtained by chemical modification and melting hyperchromicity experiments.
Biopolymers, 2014
Circular dichroism (CD) was used to assess the stabilization/destabilization imposed by oxidative lesion 7,8-dihydro-8-hydroxyadenosine (8-oxoA) on strands of RNA with different structural motifs. RNA:RNA homoduplex destabilization was observed in a position dependent manner using 10-mers as models that displayed differences between 12.7 and 15.1 C. We found that increasing the number of modifications resulted in depressed T m values of about 12-15 C per lesion. The same effect was observed on RNA:DNA heteroduplex samples. We also tested the effects of this lesion in short hairpins containing the tetraloop UUCX (X 5 A, 8-oxoA). We found that the stem was hypersensitive to substitution of A by 8-oxoA and that it destabilized the structure by >23 C. Concomitant substitution at the stem and loop prevented formation of this secondary structure or lead to other less-stable hairpins. Incorporation of this lesion at the first base of the loop had no effect on either structure. Overall, we found that the effects of 8-oxoA on RNA structure are position dependent and that its stabilization may vary from sharp decreases to small increments, in some cases, leading to the formation of other more/less stable structures. These structural changes may have larger biological implications, particularly if the oxidatively modified RNA persists, thus leading to changes in RNA reactivity and function.
Evidence for tertiary structure in natural single stranded RNAs in solution
Nucleic Acids Research, 1988
Binding Isotherms (20°C) of ethldlum bromide to a number of tRNA species at various ionic strengths indicate that I) the number n| of Intercalation sites Is high 7 to 11 per molecule, In the low salt form III, but small, 2 to 1, at high Mg 2+ or Na + when form I predominates. II) modification of tRNA at strategic positions for 3D folding prevents full expression of intercalation restriction III) maximal restriction Is obtained at salt concentrations higher than needed for full conversion to form I. It Is Inferred that restriction, which Is not observed with blhelical RNA (or DNA), requires the native tRNA 3D structure but also some physical coupling between the region of 3D folding and blhelical arms. Ribosomai RNAs, some viral RNAs, mRNA from sheep mammary gland as well as the random copolymers Poly UG, Poly AUG, Poly AUCG all exhibit Intercalation restriction. Hence 3D folding of the polyribonucleotlde chains appears to be a feature common to single-stranded RNAs when free in solution under physiological conditions.
Circular dichroism of adenine and thymine containing synthetic polynucleotides
Biopolymers, 1977
Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A‐T)]·poly[d(T‐A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double‐stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A‐t)]·poly[d(T‐A)] are due to a change in conformation in which the secondary structure goes from a C‐ to B‐type spectrum by increasing the A‐type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B‐type geometries occurs.
Probing the structure of RNAs in solution
Nucleic Acids Research, 1987
During these last years, a powerful methodology has been developed to study the secondary and tertiary structure of RNA molecules either free or engaged in complex with proteins. This method allows to test the reactivity of every nucleotide towards chemical or enzymatic probes. The detection of the modified nucleotides and RNase cleavages can be conducted by two different paths which are orientated both by the length of the studied RNA and by the nature of the probes used. The first one uses end-labeled RNA molecule and allows to detect only scissions in the RNA chain. The second approach is based on primer extension by reverse transcriptase and detects stops of transcription at modified or cleaved nucleotides. The synthesized cDNA fragments are then sized by electrophoresis on polyacrylamide:urea gels. In this paper, the various structure probes used so far are described, and their utilization is discussed. INTRODUCIION A wide range of functions are devoted to RNA molecules in the cell. RNAs are involved in all steps of protein synthesis, by storing the genetic information (messenger RNA), by participating in the structure of the mRNA decoding machinery (the ribosome), by carrying the aminoacids onto the ribosome (tRNAs). This multimolecular mechanism requires specific and coordinated RNA-RNA and RNA-protein interactions. Besides these canonical roles, several unexpected new functions for RNAs have been recently reported, e.g. enzymatic activity in RNA splicing and maturation (1-7), priming of DNA synthesis in mitochondria (8), priming reverse transcription (9), biosynthesis of the heme (10). Obviously, the three-dimensional structure of RNAs determines many of their biological activities. Thus, the precise mapping of secondary and tertiary structure features are of prime importance for a detailed understanding of the RNA functions.
Vibrational circular dichroism of A-, B-, and Z-form nucleic acids in the PO2-stretching region
Biophysical Journal, 1994
Vibrational circular dichroism (VCD) spectra were measured for H20 solutions of several natural and model DNAs (single and double strands, oligomers and polymers) in the B-form, poly(dG-dC).poly(dG-dC) in the Z-form, and various duplex RNAs in an A-form over the POstretching region. Only the symmetric POstretch at -1075 cm-' yields a significant intensity VCD signal. Differences of the POstretching VCD spectra found for these conformational types are consistent with the spectral changes seen in the base region, but no sequence dependence was seen in contrast to VCD for base modes. The B to Z transition is accompanied by an inversion of the PO VCD spectra, which is characteristic of the change in the helical sense of the nucleic acid backbone. A-RNAs give rise to the same sense of couplet VCD as do B-DNAs but have a somewhat different shape because of overlapping ribose modes. These PO-VCD spectral characteristics have been successfully modeled using simple dipole coupling calculations. The invariability of the symmetric PO stretching mode VCD spectra to the base sequence as opposed to that found for the C=O stretching and base deformation modes is evidence that this mode will provide a stable indication of the DNA helical sense.