Showing papers on "Nucleic acid secondary structure published in 1987"

Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA.

Abstract: The human U3 ribonucleoprotein (RNP) has been analyzed to determine its protein constituents, sites of protein-RNA interaction, and RNA secondary structure. By using anti-U3 RNP antibodies and extracts prepared from HeLa cells labeled in vivo, the RNP was found to contain four nonphosphorylated proteins of 36, 30, 13, and 12.5 kilodaltons and two phosphorylated proteins of 74 and 59 kilodaltons. U3 nucleotides 72-90, 106-121, 154-166, and 190-217 must contain sites that interact with proteins since these regions are immunoprecipitated after treatment of the RNP with RNase A or T1. The secondary structure was probed with specific nucleases and by chemical modification with single-strand-specific reagents that block subsequent reverse transcription. Regions that are single stranded (and therefore potentially able to interact with a substrate RNA) include an evolutionarily conserved sequence at nucleotides 104-112 and nonconserved sequences at nucleotides 65-74, 80-84, and 88-93. Nucleotides 159-168 do not appear to be highly accessible, thus making it unlikely that this U3 sequence base pairs with sequences near the 5.8S rRNA-internal transcribed spacer II junction, as previously proposed. Alternative functions of the U3 RNP are discussed, including the possibility that U3 may participate in a processing event near the 3' end of 28S rRNA.

DNA structure equilibria in the human c-myc gene

Abstract: We have employed analytical S1 nuclease analysis to identify sites with altered DNA secondary structure in the human c-myc gene. We have mapped several sites of that kind in vitro at one-base resolution but have focused our attention on one particularly stable conformational isomer which occurs approximately 270 base pairs upstream from the preferred transcription origin. We have analyzed the kinetics of that conformational equilibrium as a function of supercoil density and enzyme concentration and find that DNA structure in this region is adequately modeled as a two-state equilibrium between an undistorted (S1 nuclease insensitive) and a distorted (S1-sensitive) state. We find that at fixed supercoil density, S1 nuclease cleavage at this DNA segment can be altered in vitro by a DNA sequence change as far away as 1500 bases. We also find that the S1 nuclease cleavage at this site can be dramatically enhanced by the binding of small RNA molecules. On the basis of an analysis of S1 cutting kinetics and an analysis of DNA sequence at the S1 cleavage site, we conclude that RNA may bind directly to DNA, thereby shifting the underlying conformational equilibrium. Together, these data suggest that as a class, short RNA molecules could serve as site-specific regulatory elements in the myc gene and elsewhere.

500-MHz proton homonuclear Overhauser evidence for additional base pairs in the common arm of eukaryotic ribosomal 5S RNA: wheat germ

Abstract: A "common-arm" fragment from wheat germ (Triticum aestivum) 5S RNA has been produced by enzymatic cleavage with RNase T1 and sequenced via autoradiography of electrophoresis gels for the end-labeled fragments obtained by further RNase T1 partial digestion. The existence, base pair composition, and base pair sequence of the common arm are demonstrated for the first time by means of proton 500-MHz nuclear magnetic resonance. From Mg2+ titration, temperature variation, ring current calculations, sequence comparisons, and proton homonuclear Overhauser enhancement experiments, additional base pairs in the common arm of the eukaryotic 5S RNA secondary structure are detected. Two base pairs, G41 X C34 and A42 X U33 in the hairpin loop, could account for the lack of binding between the conserved GAAC segment of 5S RNA and the conserved Watson-Crick-complementary GT psi C segment of tRNAs.

Phage phi X174 probed by laser Raman spectroscopy: evidence for capsid-imposed constraint on DNA secondary structure.

Abstract: The Raman spectrum of the isometric bacteriophage phi X174 contains a number of well-resolved bands which have been assigned unambiguously to proteins of the capsid or to the single-stranded DNA (ssDNA) genome. Additional Raman bands of protein and DNA, which are partially overlapped in the spectrum of virus, have been resolution enhanced by Fourier deconvolution to permit improved semiquantitative measurement of spectral intensities and frequencies for structural conclusions. Raman conformation markers indicate that the ssDNA molecule within the capsid contains nucleosides of C2'-endo sugar pucker and anti-glycoside bond orientation, but the nucleic acid backbone lacks the geometry characteristic of B-form DNA. The Raman profile of encapsidated phi X DNA indicates a backbone more similar to heat-denatured DNA than to DNA containing hairpinlike secondary structure. This finding suggests limited interbase interactions in the packaged genome, which is presumably the result of constraints imposed by the viral capsid. Thus, the extensive pairing and stacking of bases indicated by Raman profiles from ssRNA viruses are not evident for the phi X174 chromosome. Overall, the proteins of the virion contain extensive beta-sheet and irregular secondary structures. Fourier deconvolution of the Raman amide I band provides an estimate of the percentage of total beta-sheet structure (approximately 60%) in all proteins of the virion. The amide III region of the spectrum confirms that beta-sheet and irregular domains are the predominant protein secondary structures. Samples of phi X174 concentrated for Raman spectroscopy by either ultracentrifugation or ultrafiltration exhibit nearly identical Raman spectra, indicating that either method can be employed to prepare intact virus without significant loss of DNA or protein components.

Graphics of RNA secondary structure; towards an object-oriented algorithm.

Abstract: We present a new algorithm for the display of RNA secondary structure. The principle of the algorithm is entirely different from those currently in use in that our algorithm is 'object oriented' while current algorithms are 'procedural'. The circular RNA molecule of chrysanthemum stunt viroid was used as input data for demonstrating the operation of the program. The major interest of this method will be found in its potential use in simulation graphics of RNA folding processes.

Isolation and characterization of nucleoprotein assembly intermediates of tobacco mosaic virus.

Abstract: During assembly of tobacco mosaic virus from pure RNA and 20S capsid protein aggregates under conditions where protein is limiting, partially assembled intermediates of specific sizes accumulate; these were isolated on sucrose density gradients. The earliest intermediate found in substantial quantity sedimented at 56 S and was shown, by measurement of its buoyant density and of the length of the RNA segment protected by the capsid protein from nuclease digestion, to consist of RNA that is 13% encapsidated (corresponding to a rod length of about 39 nm); the next intermediate sediments at 78 S and is 18% encapsidated (corresponding to a rod length of about 54 nm). Studies of the distribution of intermediates at various input ratios of protein/RNA indicated that their accumulation results from decreases in the rate constants for protein binding that are local to specific points in the course of encapsidation. After extensive nuclease digestion, the RNA still associated with the first intermediate was shown to include a portion that is unencapsidated. This segment of the RNA may be a region of stable secondary that confers the nuclease resistance despite the lack of protection by capsid protein. Such RNA secondary structure, if it exists, would also cause the accumulation of intermediates by imposing an energy barrier to subsequent rod elongation.

The Complex Cellular Networks in the Control of SV40 Gene Expression

Abstract: Electrconmcroscopical and biochemical studies have shown an association between SV40 viral proteins, viral assembly, and the biogenesis of late SV40 RNA with the complex cellular networks of the nucleus and of the cytoplasm. Pulse-chase experiments revealed a rapid transport of viral capsid proteins from the cytoplasm to the nuclear matrix, where virus assembly occurs. The nuclear matrices of SV40-infected cells retain the majority of mature SV40 virions and the rapidly labeled SV40 nuclear RNA. Isolated nuclei from SV40-infected cells when incubated in the presence of [α-32P]UTP elongate the in vivo preinitiated SV40 RNA synthesizing both long viral RNA molecules and a 94 nucleotide long promoter-proximal viral RNA species (attenuator RNA). In contrast to rapidly labeled viral nuclear RNA, the attenuator RNA is not associated with the nuclear matrix. Pretreating the cells with proflavine that interferes with RNA secondary structure increases the amount of long viral RNA molecules which become associated with the nuclear matrix. Pretreatment with DRB (1-β-ribofuranosylbenzimidazole) which enhances premature termination of RNA synthesis enhances the accumulation of the attenuator RNA in the nuclei in a DNase and salt soluble fraction which is not associated with the matrix.