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PubMed1995

Specific binding of aminoglycoside antibiotics to RNA.

BACKGROUND: Aminoglycoside antibiotics interfere with ribosomal protein synthesis and with intron splicing. Various lines of evidence suggest that RNA is the molecular target for aminoglycosides, but little is known about the recognition process. Is recognition of a particular aminoglycoside specific for certain RNA structures? If so, what are the rules for recognition? We have begun to investigate this problem by in vitro selection of RNA molecules that can specifically bind to the aminoglycoside antibiotic tobramycin. RESULTS: An RNA diversity library was used to select for sequences capable of binding to the aminoglycoside antibiotic tobramycin. After six cycles of selection, 82% of the RNA bound to tobramycin specifically. The selected RNA was reverse-transcribed into DNA, which was then cloned. At low selection stringency, an extremely large number of clones, on the order of 10(7), produced RNAs capable of binding tobramycin with Kds in the microM range (values similar to that observed for the binding of tobramycin to Escherichia coli ribosomes). Sequencing of 18 of the clones revealed no obvious consensus sequence. At higher selection stringencies (Kds in the nM range) only two consensus sequences for binding were observed. CONCLUSIONS: We have shown that RNA molecules can be readily selected that bind the aminoglycoside tobramycin. The RNAs that bind tobramycin with high affinity contain consensus binding regions that may be confined to predicted stem-loop structures. These studies open the way for understanding the basis of RNA-aminoglycoside recognition.

Anti-Bacterial Agents↗
PubMed2006

Probing the structure and function of an archaeal C/D-box methylation guide sRNA.

The genome of the hyperthermophilic archaeon Sulfolobus solfataricus contains dozens of small C/D-box sRNAs that use a complementary guide sequence to target 2'-O-ribose methylation to specific locations within ribosomal and transfer RNAs. The sRNAs are approximately 50-60 nucleotides in length and contain two RNA structural kink-turn (K-turn) motifs that are required for assembly with ribosomal protein L7Ae, Nop5, and fibrillarin to form an active ribonucleoprotein (RNP) particle. The complex catalyzes guide-directed methylation to target RNAs. Earlier work in our laboratory has characterized the assembly pathway and methylation reaction using the model sR1 sRNA from Sulfolobus acidocaldarius. This sRNA contains only one antisense region situated adjacent to the D-box, and methylation is directed to position U52 in 16S rRNA. Here we have investigated through RNA mutagenesis, the relationship between the sR1 structure and methylation-guide function. We show that although full activity of the guide requires intact C/D and C'/D' K-turn motifs, each structure plays a distinct role in the methylation reaction. The C/D motif is directly implicated in the methylation function, whereas the C'/D' element appears to play an indirect structural role by facilitating the correct folding of the RNA. Our results suggest that L7Ae facilitates the folding of the K-turn motifs (chaperone function) and, in addition, is required for methylation activity in the presence of Nop5 and Fib.

Archaeal Proteins↗
PubMed2006

L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy.

Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11(ctd)). In addition, the N-terminal domain of L11 (L11(ntd)) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11(ntd), placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11-RNA-thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11(ntd) in elongation factor binding.

Anti-Bacterial Agents↗
PubMed2000

The i-motif in nucleic acids.

Seven years after the discovery of the DNA i-motif, partial explanations for its occurrence have been uncovered, possibly involving CHellipsisO hydrogen bonds across the narrow grooves. Investigations of its biological significance have been encouraged by the demonstration and description of the intramolecular i-motif structure of human telomeric and centromeric sequences, by the recent observation of an intercalated RNA structure and by the discovery of proteins that associate with DNA sequences carrying cytosine repeats. The compatibility of the intercalation with peptide and phosphorothioate DNA analogs is favorable for possible pharmaceutical applications.

DNA↗
PubMed1978

Identification of an Escherichia coli nuclease acting on structurally altered transfer RNA molecules.

A nuclease (RNase D) that can recognize structurally altered transfer RNA molecules has been partially purified from Escherichia coli. The enzyme acts poorly on intact tRNA and is inactive with the synthetic polyribonucleotides, poly(A), poly(U), or double-stranded poly(A).poly(U). The enzyme requires Mg2+ for activity and is stimulated by the monovalent cations, K+ and NH4+. The products of the reaction are 5'-mononucleotides. The molecular weight of the protein is about 60,000 as judged by Sephadex G-100 chromatography. The enzyme does not correspond to any known E. coli ribonuclease and may represent an intracellular scavenging mechanism for denatured tRNAs and other inactive RNA molecules.

Cations↗
PubMed2006

Toward building a database of bifunctional probes for the MS3D investigation of nucleic acids structures.

This report illustrates the approaches employed to investigate critical aspects of the activity of crosslinking reagents toward nucleic acid substrates, which should be evaluated to identify candidate probes for mass spectrometric 3D (MS3D) investigations of biomolecules and macromolecular complexes. Representative members of different classes of bifunctional reagents were taken into consideration, including bikethoxal and phenyl-diglyoxal [bis-(1,2-dicarbonyls)], cisplatin (coordinative binding agents), chlorambucil and nitrogen mustard [bis-(2-chloroethyl)amines], and sym-triazine trichloride (triazines). Nanospray-Fourier transform mass spectrometry (FTMS) was applied without desalting or separation procedures to characterize the covalent products obtained by probing dinucleotide and trinucleotide substrates under a variety of experimental conditions in vitro. The carefully controlled composition of these substrates enabled us to obtain valid comparisons of probe activity toward individual nucleotides and evaluate possible base-specific effects, including the stability of the different adducts in solution under the selected reaction conditions. The gas-phase behavior of the observed products was investigated using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) to obtain valuable information for guiding the design of sequencing experiments and helping the data interpretation. Structured RNA substrates, such as HIV-1 stemloop 1, were finally employed to investigate the structural determinant of adduct formation and highlight the different nature of the spatial information provided by the various candidate probes.

Cross-Linking Reagents↗
PubMed2006

Biochemical characterization of the ribosomal decoding site.

Prior to the emergence of crystal structures of the ribosome, different ribosomal functions were identified with specific regions of ribosomal RNA by biochemical and genetic approaches. In particular, three universally conserved bases of 16S rRNA, G530, A1492 and A1493, were implicated in the interaction of the incoming aminoacyl-tRNA with the 30S subunit and mRNA. The conserved region surrounding A1492 and A1493 was called the "decoding site", based on the results of chemical probing experiments and antibiotic resistance mutations. Crystallographic studies from the Ramakrishnan laboratory have now shown that G530 loop, A1492 and A1493 undergo localized conformational changes to form an RNA structure that positions these three bases to inspect the accuracy of the codon-anticodon match with high stereochemical precision, using A-minor interactions. Some results from the pre-X-ray era may provide clues to further aspects of the decoding process.

Anticodon↗
PubMed2003

MolMovDB: analysis and visualization of conformational change and structural flexibility.

The Database of Macromolecular Movements (http://MolMovDB.org) is a collection of data and software pertaining to flexibility in protein and RNA structures. The database is organized into two parts. Firstly, a collection of 'morphs' of solved structures representing different states of a molecule provides quantitative data for flexibility and a number of graphical representations. Secondly, a classification of known motions according to type of conformational change (e.g. 'hinged domain' or 'allosteric') incorporates textual annotation and information from the literature relating to the motion, linking together many of the morphs. A variety of subsets of the morphs are being developed for use in statistical analyses. In particular, for each subset it is possible to derive distributions of various motional quantities (e.g. maximum rotation) that can be used to place a specific motion in context as being typical or atypical for a given population. Over the past year, the database has been greatly expanded and enhanced to incorporate new structures and to improve the quality of data. The 'morph server', which enables users of the database to add new morphs either from their own research or the PDB, has also been enhanced to handle nucleic acid structures and multi-chain complexes.

Computer Graphics↗
PubMed2000

Structure of the RNA inside the vesicular stomatitis virus nucleocapsid.

The structure of the viral RNA (vRNA) inside intact nucleocapsids of vesicular stomatitis virus was studied by chemical probing experiments. Most of the Watson-Crick positions of the nucleotide bases of vRNA in intact virus and in nucleoprotein (N)-RNA template were accessible to the chemical probes and the phosphates were protected. This suggests that the nucleoprotein binds to the sugar-phosphate backbone of the RNA and leaves the Watson-Crick positions free for the transcription and replication activities of the viral RNA-dependent RNA polymerase. The same architecture has been proposed for the influenza virus nucleocapsids. However, about 5% of the nucleotide bases were found to be relatively nonreactive towards the chemical probes and some bases were hyperreactive. The pattern of reactivities was the same for RNA inside virus and for RNA in N-RNA template that was purified over a CsCl gradient and which had more than 94% of the polymerase and phosphoprotein molecules removed. All reactivities were more or less equal on naked vRNA. This suggests that the variations in reactivity towards the chemical probes are caused by the presence of the nucleoprotein.

Aldehydes↗
PubMed1993

Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains.

The 5' region of HIV-1 RNA contains functional elements involved in key steps of the retroviral cycle, such as genomic RNA transcription, splicing, translation, dimerization or initiation of reverse transcription. In the present work, we investigated the conformation of the first 500 nucleotides covering the RNA leader and the 5' gag coding sequences of HIV-MAL, using chemical probing. We provide detailed information on almost each nucleotide at one of their Watson-Crick positions and on position N-7 of purines. Experiments were conducted on two in vitro transcribed RNA fragments (1 to 707 and 1 to 311). A secondary structure model was derived by combining the experimental data, computer predictions and sequence comparison. Under conditions favoring dimerization (high salt concentration), HIV-1 RNA folds into independent structural domains that can be related to defined functional regions. The first domain corresponds to TAR forming a stable stem-loop. Intrinsic structural features are found to stabilize the TAR hairpin loop. The second domain (nucleotides 56 to 299) contains the PBS sequence, which is located in a stable subdomain constrained by a four stem junction (nucleotides 139 to 218). Although the MAL isolate has an insertion near the PBS, probably resulting from the duplication of a 23-nucleotide sequence, the structural organization of this subdomain is conserved in all other HIV-1 isolates. The third domain (nucleotides 300 to 404) contains the splice donor site, packaging and dimerization elements and the AUG initiation codon of gag. A major result is the structural versatility of this region. Two mutually exclusive structures, both equally in agreement with probing data, could modulate the different functions involving this domain. The reduced accessibility of the gag translational initiation site possibly accounts for the low efficiency of the in vitro translation of the dimer. Finally, the 5' gag coding sequences form a metastable domain.

Animals↗
PubMed1983

Physical studies on a nucleoprotein from the ribosome of E. coli.

Bacterial 5S RNA and its cognate proteins constitute an attractive system to study nucleoprotein interactions. The molecular weights of the components involved are modest and they can be prepared in the quantities necessary to permit the application of material-intensive techniques like NMR and X-ray crystallography. 5S RNA is being examined by proton NMR at 500 MHz with special attention paid to the downfield NH proton region. A substantial number of assignments can be suggested in this region based on nuclear Overhauser results. The binding of protein L25 (E. coli) to the RNA gives rise to a highly characteristic set of perturbations in the spectrum of the RNA. The data suggest a localized and assignable alteration in RNA structure upon formation of the complex. In addition we have grown large crystals of RNAs related to 5S RNA and their complexes with a cognate protein. The properties of these crystals and the progress made in analyzing their structure are discussed.

Base Sequence↗
PubMed2001

PseudoBase: structural information on RNA pseudoknots.

PseudoBase is a database containing structural, functional and sequence data related to RNA pseudo-knots. It can be reached at http://wwwbio.LeidenUniv.nl/ approximately Batenburg/PKB.html. For each pseudoknot, thirteen items are stored, for example the relevant sequence, the stem positions of the pseudoknot, the EMBL accession number of the sequence and the support that can be given regarding the reliability of the pseudo-knot. Since the last publication, information on sizes of the stems and the loops in the pseudoknots has been added. Also added are alternative entries that produce surveys of where the pseudoknots are, sorted according to stem size or loop size.

Base Sequence↗
PubMed2005

Effects of nucleotide substitution and modification on the stability and structure of helix 69 from 28S rRNA.

The helix 69 (H69) region of the large subunit (28S) rRNA of Homo sapiens contains five pseudouridine (Psi) residues out of 19 total nucleotides (26%), three of which are universally or highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine. The role of a loop nucleotide substitution from A in bacteria (position 1918 in Escherichia coli 23S rRNA) to G in eukaryotes (position in 3734 in H. sapiens) was also examined. The thermodynamic parameters were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by 1H NMR and circular dichroism spectroscopy. In addition, a [1,3-15N]Psi phosphoramidite was used to generate H69 analogs with site-specific 15N labels. By using this approach, different Psi residues can be clearly distinguished from one another in 1H NMR experiments. The effects of pseudouridine on H. sapiens H69 are consistent with previous studies on tRNA, rRNA, and snRNA models in which the nucleotide offers stabilization of duplex regions through PsiN1H-mediated hydrogen bonds. The overall secondary structure and base-pairing patterns of human H69 are similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved. Nonetheless, pseudouridine-containing RNAs have subtle differences in their structures and stabilities compared to the corresponding uridine-containing analogs, suggesting possible roles for Psi such as maintaining translation fidelity.

Circular Dichroism↗
PubMed2004

Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase.

Group II intron-encoded proteins (IEPs) have both reverse transcriptase (RT) activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active RNA structure. The LtrA protein encoded by the Lactococcus lactis Ll.LtrB group II intron contains an N-terminal RT domain, with conserved sequence motifs RT1 to 7 found in the fingers and palm of retroviral RTs; domain X, associated with maturase activity; and C-terminal DNA-binding and DNA endonuclease domains. Here, partial proteolysis of LtrA with trypsin and Arg-C shows major cleavage sites in RT1, and between the RT and X domains. Group II intron and related non-LTR retroelement RTs contain an N-terminal extension and several insertions relative to retroviral RTs, some with conserved features implying functional importance. Sequence alignments, secondary-structure predictions, and hydrophobicity profiles suggest that domain X is related structurally to the thumb of retroviral RTs. Three-dimensional models of LtrA constructed by "threading" the aligned sequence on X-ray crystal structures of HIV-1 RT (1) account for the proteolytic cleavage sites; (2) suggest a template-primer binding track analogous to that of HIV-1 RT; and (3) show that conserved regions in splicing-competent LtrA variants include regions of the RT and X (thumb) domains in and around the template-primer binding track, distal regions of the fingers, and patches on the protein's back surface. These regions potentially comprise an extended RNA-binding surface that interacts with different regions of the intron for RNA splicing and reverse transcription.

Amino Acid Sequence↗
PubMed1996

Nucleic acid crystallography: a view from the nucleic acid database.

What are the future directions of the field of nucleic acid crystallography? Although there have been many duplex structures determined, the sample is still relatively small. This is especially true if one wants to derive enough information about the relationships between sequence and structure. Indeed, there are data for all the possible 10 dimer steps, but for some steps it is very limited. If the structural code resides in trimers or tetrad steps then there is simply not enough data to do meaningful statistical analyses. So the first direction that needs to be explored is the determination of more structures with more varied sequences. The other noticeable thing about the data is the shortness of the strands. While it is probably true that attempts to crystallize very long sequences will not meet with success, the idea of crystallizing sequences engineered to fit together via sticky ends such as has been done for the CAP-DNA complex (Schultz et al., 1990) should give data about the behavior of much longer stretches of DNA. The question of the effects of environment on the structure of DNA continues to be a very important one to address since DNA is rarely alone. The preliminary data we have analysed from the current sample shows that the conformation of some steps are very sensitive to packing type. Numerous studies of the hydration around DNA shows that there is a real synergy between the hydration structure and the base conformation. More data will allow further quantitation of these observations. RNA structure is the next very exciting frontier. The emerging structures of duplexes with internal loops, the two hammerhead ribozyme structures and the group I intron ribozyme have given us a glimpse of the complexity and elegance of this class of molecules. With the technology now in place to allow the determination of the structures of these molecules, the expectation is that now we will see a large increase in the number of these structures in the NDB.

Base Sequence↗
PubMed2002

Coenzymes as coribozymes.

Coenzymes are small organic molecules that supply a varied set of reactive groups to protein enzymes, thereby diversifying catalysis beyond the chemistries of amino acid sidechains. As RNA structures begin with a more limited chemical diversity than proteins, it seems likely that RNA enzymes would also use functional groups from other molecules to support a complex RNA world metabolism. In fact, ribonucleotide moieties in many coenzymes have long been thought to be surviving vestiges of covalently bound coenzymes in an RNA world. The idea of coenzyme utilization by ribozymes can be explored by selection-amplification of coenzyme-binding RNAs and coenzyme-assisted ribozymes. Here, we review coenzyme-RNAs, and discuss their possible significance for RNA-mediated metabolism. In summary, a plausible route from prebiotic chemistry to ribozyme biochemistry exists for CoA, and via similar activities, likely exists for all the nucleotidyl coenzymes.

Base Sequence↗
PubMed2003

The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA.

The HIV type 1 (HIV-1) Tat protein stimulates transcription elongation by recruiting P-TEFb (CDK9/cyclin T1) to the transactivation response (TAR) RNA structure. Tat-induced CDK9 kinase has been shown to phosphorylate Ser-5 of RNA polymerase II (RNAP II) C-terminal domain (CTD). Results presented here demonstrate that Tat-induced Ser-5 phosphorylation of CTD by P-TEFb stimulates the guanylyltransferase activity of human capping enzyme and RNA cap formation. Sequential phosphorylation of CTD by Tat-induced P-TEFb enhances the stimulation of human capping enzyme guanylyltransferase activity and RNA cap formation by transcription factor IIH-mediated CTD phosphorylation. Using an immobilized template assay that permits isolation of transcription complexes, we show that Tat/TAR-dependent phosphorylation of RNAP II CTD stimulates cotranscriptional capping of HIV-1 mRNA. Upon transcriptional induction of latently infected cells, accumulation of capped transcripts occurs along with Ser-5-phosphorylated RNAP II in the promoter proximal region of the HIV-1 genome. Therefore, these observations suggest that Tat/TAR-dependent phosphorylation of RNAP II CTD is crucial not only in promoting transcription elongation but also in stimulating nascent viral RNA capping.

Binding Sites↗
PubMed1979

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25.

An RNA fragment, constituting three subfragments of nucleotide sequences 1-11, 69-87 and 89-120, is the most ribonuclease-resistant part of the native 5S RNA of Escherichia coli, at 0 degrees C. A smaller fragment of nucleotide sequence 69-87 and 90-110 is ribonuclease-resistant at 25 degrees. Degradation of the L25-5S RNA complex with ribonuclease A or T2 yielded RNA fragments similar to those of the free 5S RNA at 0 degrees C and 25 degrees C; moreover L25 remained strongly bound to both RNA fragments and also produced some opening of the RNA structure in at least two positions. Protein L18 initially protected most of the 5S RNA against ribonuclease digestion, at 0 degrees C, but was then gradually released prior to the formation of the larger RNA fragment. It cannot be concluded, therefore, as it was earlier (Gray et al., 1973), that this RNA fragment contains the primary binding site of L18.

Base Sequence↗