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At least 1,477 records · Page 82Linked to original sources
PubMed1999

Crystal structure of a conserved ribosomal protein-RNA complex.

The structure of a highly conserved complex between a 58-nucleotide domain of large subunit ribosomal RNA and the RNA-binding domain of ribosomal protein L11 has been solved at 2.8 angstrom resolution. It reveals a precisely folded RNA structure that is stabilized by extensive tertiary contacts and contains an unusually large core of stacked bases. A bulge loop base from one hairpin of the RNA is intercalated into the distorted major groove of another helix; the protein locks this tertiary interaction into place by binding to the intercalated base from the minor groove side. This direct interaction with a key ribosomal RNA tertiary interaction suggests that part of the role of L11 is to stabilize an unusual RNA fold within the ribosome.

Amino Acid Sequence↗
PubMed2004

On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites.

Viroids and most viral satellites have small, noncoding, and highly structured RNA genomes. How they cause disease symptoms without encoding proteins and why they have characteristic secondary structures are two longstanding questions. Recent studies have shown that both viroids and satellites are capable of inducing RNA silencing, suggesting a possible role of this mechanism in the pathology and evolution of these subviral RNAs. Here we show that preventing RNA silencing in tobacco, using a silencing suppressor, greatly reduces the symptoms caused by the Y satellite of cucumber mosaic virus. Furthermore, tomato plants expressing hairpin RNA, derived from potato spindle tuber viroid, developed symptoms similar to those of potato spindle tuber viroid infection. These results provide evidence suggesting that viroids and satellites cause disease symptoms by directing RNA silencing against physiologically important host genes. We also show that viroid and satellite RNAs are significantly resistant to RNA silencing-mediated degradation, suggesting that RNA silencing is an important selection pressure shaping the evolution of the secondary structures of these pathogens.

Base Sequence↗
PubMed2006

Structural basis for altering the stability of homologous RNAs from a mesophilic and a thermophilic bacterium.

Tertiary RNA structures from thermophilic bacteria generally are more stable than their mesophilic homologs. To understand the structural basis of the increase in stability, we investigated equilibrium folding of the specificity domain (S-domain) of RNase P RNA from a mesophilic (Escherichia coli) and a thermophilic (Thermus thermophilus) bacterium. Equilibrium folding of both S-domains is described by a minimal, three-state folding scheme, U-to-I-to-N. In the I-to-N transition of the thermophilic S-domain, more structure forms and protections are stronger against T1 nuclease and hydroxyl radical reactions. Phylogenetic comparison in the context of the native structure reveals that among 39 nucleotide differences between these S-domains, 12 likely contribute to higher stability. These residues participate in extensive networks of hydrogen bonding, stacking, and metal ion coordination throughout the molecule. The thermophilic S-domain achieves higher stability by mutating strategic base pairs to G-C, decreasing surface accessibility of the native state, and increasing the amount of structure formation in the native folding transition. An E. coli S-domain mutant containing these 12 nt has the same stability and folding cooperativity as the T. thermophilus S-domain. E. coli S-domain mutants containing a subset of 4 or 6 nt have the same stability as the T. thermophilus S-domain but the same folding cooperativity as the E. coli S-domain. These results show that increasing stability can be accomplished by mutations within a local structure, but increasing folding cooperativity needs concerted changes among multiple structural units.

Base Sequence↗
PubMed2000

Recognition of a cognate RNA aptamer by neomycin B: quantitative evaluation of hydrogen bonding and electrostatic interactions.

Aminoglycosides are an important class of antibiotic that selectively target RNA structural motifs. Recently we have demonstrated copper derivatives of amino-glycosides to be efficient cleavage agents for cognate RNA motifs. To fully develop their potential as pharmaceutical agents it is necessary to understand both the structural mechanisms used by aminoglycosides to target RNA, and the relative contributions of hydrogen bonding and electrostatic interactions to recognition selectivity. Herein we report results from a calorimetric analysis of a stem-loop 23mer RNA aptamer complexed to the aminoglycoside neomycin B. Key thermodynamic parameters for complex formation have been determined by isothermal titration calorimetry, and from the metal-ion dependence of these binding parameters the relative contributions of electrostatics and hydrogen bonding toward binding affinity have been assessed. The principal mechanism for recognition and binding of neomycin B to the RNA major groove is mediated by hydrogen bonding.

Base Sequence↗
PubMed1976

Secondary structure of ovalbumin messenger RNA.

The secondary structure of highly purified ovalbumin mRNA was studied by automated thermal denaturation techniques and the data were subjected to computer processing. Comparative studies with 20 natural and synthetic model nucleic acids suggested that the secondary structure of ovalbumin mRNA possesses the following features: the extent of base pairing of ovalbumin mRNA is similar to that found in tRNAs or ribosomal RNAs; the secondary structure of ovalbumin mRNA is more thermolabile than any of the model compounds tested, including the copolymer poly(A-U); ovalbumin mRNA does not have extensive G-C rich stems as found in tRNAs or ribosomal RNAs; the base composition of the double-stranded regions reveals 54% G-C residues which was significantly higher than that noted in the whole molecule (approximately 41.5% G-C). The presence of 46% A-U pairs in short stems of about five base pairs would have a very large destabilizing effect on the secondary structure of ovalbumin mRNA. However, at 0.175 M monovalent cations and 36 degrees C most of the secondary structure of ovalbumin mRNA is preserved. These data suggest that the double-stranded regions in ovalbumin mRNA are of sufficient length to provide the necessary stability for maintaining the open loop regions in an appropriate conformation which may be required for the biological function of ovalbumin mRNA. Furthermore, the lability of the double-stranded regions in ovalbumin mRNA may also be important for the biological function of this mRNA.

Base Sequence↗
PubMed1993

The interaction between the iron-responsive element binding protein and its cognate RNA is highly dependent upon both RNA sequence and structure.

To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.

Base Sequence↗
PubMed1995

Ribonucleosides and RNA.

Landmark discoveries such as the autocatalytic cleavage activity of certain RNA molecules, as well as small oligoribonucletide ribozymes and later the in vitro evolution of novel bioactive oligoribonucleotides (SELEX), have created entire new fields of biochemical research. The discovery of SELEX has provided a method for producing high-affinity nucleic acid ligands with high binding specificity to important medicinal targets. Including modified nucleotides into RNA ligands derived from SELEX may yield improved RNA therapeutics. The chemistry of oligoribonucleotides in comparison to oligodeoxyribonucleotides has led to resurgent attention on the role of modified nucleotides in RNA structure and function. Such modifications are also employed to impart stability towards endonuclease degradation on oligoribonucleotides.

Base Sequence↗
PubMed2007

Molecular aspects on the interaction of protoberberine, benzophenanthridine, and aristolochia group of alkaloids with nucleic acid structures and biological perspectives.

Alkaloids occupy an important position in chemistry and pharmacology. Among the various alkaloids, berberine and coralyne of the protoberberine group, sanguinarine of the benzophenanthridine group, and aristololactam-beta-d-glucoside of the aristolochia group have potential to form molecular complexes with nucleic acid structures and have attracted recent attention for their prospective clinical and pharmacological utility. This review highlights (i) the physicochemical properties of these alkaloids under various environmental conditions, (ii) the structure and functional aspects of various forms of deoxyribonucleic acid (DNA) (B-form, Z-form, H(L)-form, protonated form, and triple helical form) and ribonucleic acid (RNA) (A-form, protonated form, and triple helical form), and (iii) the interaction of these alkaloids with various polymorphic DNA and RNA structures reported by several research groups employing various analytical techniques like absorbance, fluorescence, circular dichroism, and NMR spectroscopy; electrospray ionization mass spectrometry, thermal melting, viscosity, and DNase footprinting as well as molecular modeling and thermodynamic studies to provide detailed binding mechanism at the molecular level for structure-activity relationship. Nucleic acids binding properties of these alkaloids are interpreted in relation to their biological activity.

Alkaloids↗
PubMed2005

On the structural features of hairpin triloops in rRNA: from nucleotide to global conformational change upon ligand binding.

RNA structure can be viewed as both a construct composed of various structural motifs and a flexible polymer that is substantially influenced by its environment. In this light, the present paper represents an attempt to reconcile the two standpoints. By using the 3D structures both of four (16S and 23S) portions of unbound 50S, H50S, and T30S ribosomal subunits and of 38 large ribonucleoligand complexes as the starting point, the behavior, which is induced by ligand binding, of 73 hairpin triloops with closing g-c and c-g base pairs was investigated using root-mean-square deviation (RMSD) approach and pseudotorsional (eta,theta) convention at the nucleotide-by-nucleotide level. Triloops were annotated in accordance with a recent proposal of geometric nomenclature. A simple measure for the determination of the strain of a triloop is introduced. It is believed that a possible classification of the interior triloops, based on the 2D eta-theta unique path, will aid to conceive their local behavior upon ligand binding. All rRNA residues in contact with ligands as well as regions of considerable conformational changes upon complex formation were identified. The analysis offers the answer to: how proximal to and how far from the actual ligand-binding sites the structural changes occur?

Base Sequence↗
PubMed1998

Crystal structure of an RNA octamer duplex r(CCCIUGGG)2 incorporating tandem I.U wobbles.

The crystal structure of the RNA octamer duplex r(CCCIUGGG)2has been elucidated at 2.5 A resolution. The crystals belong to the space group P21and have unit cell constants a = 33.44 A, b = 43.41 A, c = 49.39 A and beta = 104.7 degrees with three independent duplexes (duplexes 1-3) in the asymmetric unit. The structure was solved by the molecular replacement method and refined to an Rwork/Rfree of 0.185/0.243 using 3765 reflections between 8.0 and 2.5 A. This is the first report of an RNA crystal structure incorporating I.U wobbles and three molecules in the asymmetric unit. Duplex 1 displays a kink of 24 degrees between the mismatch sites, while duplexes 2 and 3 have two kinks each of 19 degrees and 27 degrees, and 24 degrees and 29 degrees, respectively, on either side of the tandem mismatches. At the I.U/U.I mismatch steps, duplex 1 has a twist angle of 33.9 degrees, close to the average for all base pair steps, but duplexes 2 and 3 are underwound, with twist angles of 24.4 degrees and 26.5 degrees, respectively. The tandem I.U wobbles show intrastrand purine-pyrimidine stacking but exhibit interstrand purine-purine stacking with the flanking C.G pairs. The three independent duplexes are stacked non-coaxially in a head-to-tail fashion to form infinite pseudo-continuous helical columns which form intercolumn hydrogen bonding interactions through the 2'-hydroxyl groups where the minor grooves come together.

Base Pairing↗
PubMed2001

Detection of antisense and ribozyme accessible sites on native mRNAs: application to NCOA3 mRNA.

The efficacies of antisense oligonucleotides and ribozymes are greatly dependent on the accessibility of their mRNA targets. Target site accessibility is affected by both RNA structure and the proteins associated along the length of the RNA. To mimic the native state of mRNA for site identification, we have previously used endogenous mRNAs in cellular extracts as targets for defined sequence oligodeoxynucleotides (ODNs) designed to identify both antisense pairing and potential ribozyme cleavage sites. The rationale for this approach is that the specific pairing of an ODN with a mRNA forms a DNA:RNA hybrid that is cleaved by the endogenous RNaseH in the cell extract. To extend the usefulness of this basic approach, we report here the use of semi-random ODN libraries to identify hammerhead ribozyme cleavage sites. Thus, the most accessible sites for antisense and ribozyme base pairing are selected by this approach. A novel feature of the approach described here is the use of terminal transferase-dependent PCR (TDPCR) after reverse transcription to estimate the cleavage efficiency and to precisely determine the RNaseH and ribozyme cleavage sites on mRNAs in cell extracts following treatment with ODN or ribozyme libraries. As a model system, we have targeted the NCOA3 (also known as AIB-1) mRNA in cell extracts. The NCOA3 mRNA encodes a nuclear receptor co-activator that is amplified and over-expressed in a high proportion of breast and ovarian cancers. A highly accessible site on this mRNA was identified, and a ribozyme targeted to this site was demonstrated to effectively downregulate NCOA3 function in cells.

Base Pairing↗
PubMed1995

Novel three-dimensional 1H-13C-31P triple resonance experiments for sequential backbone correlations in nucleic acids.

Backbone-driven assignment methods that utilize covalent connectivities have greatly facilitated spectral assignments of proteins. In nucleic acids, 1H-13C-31P correlations could play a similar role, and several related experiments (HCP) have recently been presented for backbone-driven sequential assignments in RNA. The three-dimensional extension of 1H-31P Het-Cor (P,H-COSY-H,C-HMQC) and Het-TOCSY (P,H-TOCSY-H,C-HMQC) experiments presented here complements HCP experiments as tools for spectral assignments and extraction of dihedral angle constraints. By relying on 1H-31P rather than 13C-31P couplings to generate cross peaks, the strongest connectivities are observed in different spectral regions, increasing the likelihood of resolving spectral overlap. In addition, semiquantitative estimates of 1H-31P and 13C-31P couplings provide dihedral angle constraints for RNA structure determination.

Base Sequence↗
PubMed1995

Unusual folding regions and ribosome landing pad within hepatitis C virus and pestivirus RNAs.

A statistically significant folding region is identified in the 5' untranslated region (5'-UTR) of hepatitis C virus (HCV), bovine viral diarrhea virus and hog cholera virus. This unusual folding region (UFR) detected in HCV encompasses 199 nucleotides (nt) and coincides with the reported internal ribosome entry site or ribosome landing pad (RLP), as determined by the 5' and 3' deletions [Tsukiyama-Kohara et al., J. Virol. 66 (1992) 1476-1483]. The RNA structure predicted in the UFR of HCV consists of a large stem-loop and a pseudoknot. The proposed structural model is consistent with RNase sensitivity studies [Brown et al., Nucleic Acids Res. 20 (1992) 5041-5045]. Moreover, the structure is highly conserved among these divergent HCV and pestivirus RNAs. The covariation of paired bases in the helical regions offers support for the proposed structural models. The pseudoknot predicted in these UFR shares a similar structural feature to those proposed in the RLP of cardioviruses, aphthoviruses and hepatitis A virus. Based on the common structural motif, a putative base-pairing model between HCV RNA and 18S rRNA, as well as pestiviral RNAs and 18S rRNA are suggested. Intriguingly, the proposed base-pairing models in this study are comparable to those proposed in picornaviruses in terms of their folded shape and location of the predicted complementary sequences between viral RNAs and 18S rRNA. Taken together, we suggest that the common base-pairing model between the UFR detected in the 5'-UTR of pestivirus and HCV and 18S rRNA have a general function in the internal initiation of cap-independent translation.

Base Sequence↗
PubMed2005

Translational enhancement of HCV RNA genotype 1b by 3'-untranslated and envelope 2 protein-coding sequences.

HCV RNA has a unique regulatory mechanism for translation. The X region of 3'-UTR and core-coding sequence regulate HCV translation. In this study, we clarified that the entire 3'-UTR also enhances HCV translation, and the envelope-coding sequence of HCV genotype 1b increases degree of this enhancement. In the luciferase reporter assay using rabbit reticulocyte lysates, translational enhancement by 3'-UTR with core to E2 regions was 25-fold higher when compared with control RNA lacking the 3'-UTR. Presence of the entire E2 sequence was important for this enhancement. This phenomenon was not due to transcript stability, and envelope protein alone did not affect translation. E2-coding sequence of genotype 1a had no effect on translation. We observed the same results in animal cell culture systems using bicistronic RNA. Structural protein-coding sequences and 3'-UTR of HCV RNA regulate viral translation, and a target for antiviral agents may be present in these regions.

3' Untranslated Regions↗
PubMed1999

mRNA degradation. A tale of poly(A) and multiprotein machines.

The Escherichia coli RNA degradosome is a multiprotein complex containing an endoribonuclease, polynucleotide phosphorylase and a DEAD-box RNA helicase. A related complex has been described in the spinach chloroplast. The exosome and the mtEXO complex have recently been described in yeast and it is likely that related complexes also exist in animal cells. This research suggests the widespread existence of sophisticated machines for the efficient degradation of messenger RNA. The DEAD-box helicase in the degradosome can unwind regions of RNA structure that interfere with 3'-5' degradation. The polyadenylation of RNA 3' ends is also known to promote degradation by creating a 'toehold' for the degradation machinery. Much remains to be learned about the regulation of mRNA stability. The complexity of the degradation process, both in the eubacteria and in the eukaryotes, suggests that many steps are possible points of control.

Bacterial Proteins↗
PubMed2003

Substrate analogues for an RNA-editing adenosine deaminase: mechanistic investigation and inhibitor design.

ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that occur in eukaryotic mRNAs, including the mRNAs of glutamate and serotonin receptors. ADARs capable of editing biologically relevant RNA substrates have been identified. In addition, the consequence of the RNA-editing reaction on the function of the gene product is known in several cases. However, our understanding of the chemical mechanism of the ADAR-catalyzed adenosine deamination in RNA is lagging. By studying analogues of a naturally occurring substrate for ADAR2, we infer features of the enzyme's active site and reaction mechanism. 8-Aza substitution at adenosine in various RNA substrates accelerates the rate of deamination at these sites by ADAR2 (2.8-17-fold). The magnitude of this "aza effect" depends on the RNA structural context of the reacting nucleotide. N(6)-Methyladenosine in RNA is a slow substrate for ADAR2 (rate is 2% that of adenosine), with no product observed with N(6)-ethyladenosine, suggesting a limited size of the leaving group pocket. 2,6-Diaminopurine ribonucleoside in RNA is not a substrate for ADAR, in contrast to adenosine deaminase (ADA), which catalyzes a similar reaction on nucleosides. This and other results indicate that ADAR2 uses a base recognition strategy different from that of ADA. Consistent with the large 8-aza effect observed for the ADAR2 reaction, we find that 8-azanebularine, as the free nucleoside, inhibits the ADAR2 reaction (IC(50) = 15 +/- 3 mM) with no inhibition observed with nebularine or coformycin.

Adenosine↗
PubMed2001

MUSTA--a general, efficient, automated method for multiple structure alignment and detection of common motifs: application to proteins.

Here we present an algorithm designed to carry out multiple structure alignment and to detect recurring substructural motifs. So far we have implemented it for comparison of protein structures. However, this general method is applicable to comparisons of RNA structures and to detection of a pharmacophore in a series of drug molecules. Further, its sequence order independence permits its application to detection of motifs on protein surfaces, interfaces, and binding/active sites. While there are many methods designed to carry out pairwise structure comparisons, there are only a handful geared toward the multiple structure alignment task. Most of these tackle multiple structure comparison as a collection of pairwise structure comparison tasks. The multiple structural alignment algorithm presented here automatically finds the largest common substructure (core) of atoms that appears in all the molecules in the ensemble. The detection of the core and the structural alignment are done simultaneously. The algorithm begins by finding small substructures that are common to all the proteins in the ensemble. One of the molecules is considered the reference; the others are the source molecules. The small substructures are stored in special arrays termed combinatorial buckets, which define sets of multistructural alignments from the source molecules that coincide with the same small set of reference atoms (C(alpha)-atoms here). These substructures are initial small fragments that have congruent copies in each of the proteins. The substructures are extended, through the processing of the combinatorial buckets, by clustering the superpositions (transformations). The method is very efficient.

Algorithms↗