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At least 1,261 records · Page 70Linked to original sources
PubMed2006

Structural biology of RNA polymerase III: subcomplex C17/25 X-ray structure and 11 subunit enzyme model.

We obtained an 11 subunit model of RNA polymerase (Pol) III by combining a homology model of the nine subunit core enzyme with a new X-ray structure of the subcomplex C17/25. Compared to Pol II, Pol III shows a conserved active center for RNA synthesis but a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes a HRDC domain in subunit C17 that is translated by 35 A and rotated by 150 degrees compared to its Pol II counterpart. The HRDC domain is essential in vivo, folds independently in vitro, and, unlike other HRDC domains, shows no indication of nucleic acid binding. Thus, the HRDC domain is a functional module that could account for the role of C17 in Pol III promoter-specific initiation. During elongation, C17/25 may bind Pol III transcripts emerging from the adjacent exit pore, because the subcomplex binds to tRNA in vitro.

Amino Acid Sequence↗
PubMed1995

A method for predicting common structures of homologous RNAs.

We have developed a procedure, composed of a set of computer programs, for predicting common RNA structures of homologous sequences. Given a set of homologous RNAs, these programs perform a multiple sequence alignment, generate a list of possible helical stems that are thermodynamically favored in RNA folding from a selected individual sequence, establish a conserved stem list by inspecting the equivalent base pairings and/or conserved helical stems from the derived alignment of homologous RNAs, and build common RNA secondary structures with the maximum scores (i.e., compensatory base changes and number of base pairs, etc.). The approach is a combination of phylogenetic and thermodynamic methods and has been applied to the prediction of common folding structures of the 5' untranslated regions in a number of positive RNA viruses.

Algorithms↗
PubMed2005

A novel cGUUAg tetraloop structure with a conserved yYNMGg-type backbone conformation from cloverleaf 1 of bovine enterovirus 1 RNA.

The 5'-terminal cloverleaf (CL)-like RNA structures are essential for the initiation of positive- and negative-strand RNA synthesis of entero- and rhinoviruses. SLD is the cognate RNA ligand of the viral proteinase 3C (3C(pro)), which is an indispensable component of the viral replication initiation complex. The structure of an 18mer RNA representing the apical stem and the cGUUAg D-loop of SLD from the first 5'-CL of BEV1 was determined in solution to a root-mean-square deviation (r.m.s.d.) (all heavy atoms) of 0.59 A (PDB 1Z30). The first (antiG) and last (synA) nucleotide of the D-loop forms a novel 'pseudo base pair' without direct hydrogen bonds. The backbone conformation and the base-stacking pattern of the cGUUAg-loop, however, are highly similar to that of the coxsackieviral uCACGg D-loop (PDB 1RFR) and of the stable cUUCGg tetraloop (PDB 1F7Y) but surprisingly dissimilar to the structure of a cGUAAg stable tetraloop (PDB 1MSY), even though the cGUUAg BEV D-loop and the cGUAAg tetraloop differ by 1 nt only. Together with the presented binding data, these findings provide independent experimental evidence for our model [O. Ohlenschlager, J. Wohnert, E. Bucci, S. Seitz, S. Hafner, R. Ramachandran, R. Zell and M. Gorlach (2004) Structure, 12, 237-248] that the proteinase 3C(pro) recognizes structure rather than sequence.

3C Viral Proteases↗
PubMed1993

An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation.

Selective encapsidation of hepatitis B virus (HBV) genomic RNA within cytoplasmic core particles requires recognition of the cis-encapsidation signal, (termed epsilon) located at the 5' end of genomic RNA. By transfecting plasmids expressing chimeric RNAs bearing HBV sequences fused to lacZ, we have mapped the minimal region of epsilon to the 5' 94 nucleotides (nt) of genomic RNA. Enzymatic probing of the RNA secondary structure in this region (by using either in vitro transcripts or RNA extracted from HBV core particles) reveals a stem-loop structure containing a lower stem, a 6-nt bulge, an upper stem with a single unpaired U residue, and a 6-nt loop. The functional role of this structure in encapsidation was explored by examining the effects of mutations in epsilon on encapsidation of RNA in vivo. These studies reveal that (i) in the lower stem, base pairing but not specific primary sequence is required for function; (ii) there is no requirement for base pairing in the lower portion of the upper stem, but base pairing elsewhere in this stem contributes to packaging efficiency; (iii) the presence of the 6-nt bulge, but not its primary sequence, is important for function; and (iv) specific nucleotide sequences in the loop and in regions of the upper stem are critical for RNA encapsidation.

Base Sequence↗
PubMed2006

Evaluation of the dynamic structure of DsrA RNA from E. coli and its functional consequences.

DsrA RNA is an 87-nucleotide regulatory non-protein-coding RNA of Escherichia coli for which two secondary structure models (I and II) have been proposed. We have compared these models by the energy calculations, which revealed that the currently accepted model II should be rejected on the basis of thermodynamics. Here we provide new results of nuclease footprinting analysis and the application of RNA technologies that have not previously been used for DsrA RNA structural studies, such as hydrolysis with RNase H, DNAzyme, hydroxyl radicals and lead. These approaches together with bioinformatics calculations provided strong arguments for a new model III. This model clearly shows that the long U-rich region between hairpins 1 and 2 is double-stranded. These findings shed new light on DsrA RNA-Hfq interactions.

Computational Biology↗
PubMed1999

The stem hairpin loop structure of p2Sp1 RNA is required for RNA-cleaving activity.

We studied the hairpin-loop structure of an RNA fragment (GUUUCGUACAAAC) (R13) with the sequence corresponding to the self-cleavage domain in the precursor of an RNA molecule from bacteriophage T4-infected Escherichia coli cells (p2Sp1 RNA). In order to determine the influence of the hairpin-loop structure on these sequence-specific cleavage reactions, we have synthesized oligoribonucleotides containing hairpin-loop, double-helical stem-loop, and single-stranded RNA structures. The cleavage was affected by the hairpin-loop structure. Furthermore, the helix-stem, which retains the thermodynamically extrastable stem hairpin-loop structures, is also important for the cleavage activity. However, the thermodynamically extrastable helix-stem structure reduced the cleavage activity of the adjacent UA and CA sequences at the helix-stem site. For the cleavage reactions of the RNA cleavage products, the R6 (ACAAAC), R7 (GUUUCGU), and R9 (GUUUCGUAC) mers from the parent RNA, R13 (GUUUCGUACAAAC), a very slight amount of cleavage product (2%) from the RNA 9 was observed, but no reaction occurred for the R6 and R7. We also describe the influences of the sequences (UA and CA) on the cleavage activity.

Base Sequence↗
PubMed1997

Design and analysis of molecular motifs for specific recognition of RNA.

Selective targeting of RNA has become a recent priority in drug design strategies due to the emergence of retroviruses, the need for new antibiotics to counter drug resistance, and our increased awareness of the essential role RNA and RNA structures play in the progression of disease. Most organic compounds known to specifically target RNA are complex, naturally occurring antibiotics that are difficult to synthesize or derivatize and modification of these compounds to optimize interactions with structurally unique RNAs is difficult. The de novo design of synthetically accessible analogues is one possible alternative; however, little is known about the RNA recognition principles on which to design new compounds and limited information on RNA structure in general is available. To contribute to the growing body of knowledge on RNA recognition principles, we have prepared two series of polycationic RNA-binding agents, one with a linear scaffold, the other with a macrocyclic scaffold. We evaluated these compounds for their ability to bind to DNA and RNA, as well as to a specific RNA, the regulatory sequence, RRE, derived from HIV-1, by using thermal melting, circular dichroism, and electrophoresis gel shift methods. Out results suggest that cationic charge centers of high pKa that are displayed along a scaffold of limited flexibility bind preferentially to RNA, most likely within the major groove. Related derivatives that bind more strongly to DNA more closely mimic classical DNA minor-groove binding agents. Several of the macrocyclic polycations expand on a new binding motif where purine bases in duplex RNA are complexed within the macrocyclic cavity, enhancing base-pair opening processes and ultimately destabilizing the RNA duplex. The results in this report should prove a helpful addition to the growing information on molecular motifs that specifically bind to RNA.

Amino Acid Sequence↗
PubMed2006

Structure of eIF3b RNA recognition motif and its interaction with eIF3j: structural insights into the recruitment of eIF3b to the 40 S ribosomal subunit.

Mammalian eIF3 is a 700-kDa multiprotein complex essential for initiation of protein synthesis in eukaryotic cells. It consists of 13 subunits (eIF3a to -m), among which eIF3b serves as a major scaffolding protein. Here we report the solution structure of the N-terminal RNA recognition motif of human eIF3b (eIF3b-RRM) determined by NMR spectroscopy. The structure reveals a noncanonical RRM with a negatively charged surface in the beta-sheet area contradictory with potential RNA binding activity. Instead, eIF3j, which is required for stable 40 S ribosome binding of the eIF3 complex, specifically binds to the rear alpha-helices of the eIF3b-RRM, opposite to its beta-sheet surface. Moreover, we identify that an N-terminal 69-amino acid peptide of eIF3j is sufficient for binding to eIF3b-RRM and that this interaction is essential for eIF3b-RRM recruitment to the 40 S ribosomal subunit. Our results provide the first structure of an important subdomain of a core eIF3 subunit and detailed insights into protein-protein interactions between two eIF3 subunits required for stable eIF3 recruitment to the 40 S subunit.

Amino Acid Motifs↗
PubMed1999

RNA as a small-molecule drug target: doubling the value of genomics.

Recent advances in the determination of RNA structure and function have led to new opportunities that will have a significant impact on the pharmaceutical industry. RNA, which, among other functions, serves as a messenger between DNA and proteins, was thought to be an entirely flexible molecule without significant structural complexity. However, recent studies have revealed a surprising intricacy in RNA structure. This observation unlocks opportunities for the pharmaceutical industry to target RNA with small molecules. Because both proteins and their specific mRNAs are potential drug-binding sites, the number of targets revealed from genome sequencing efforts is effectively doubled. Perhaps more importantly, drugs that bind to RNA might produce effects that cannot be achieved by drugs that bind to proteins.

Journal Article↗
PubMed2005

Structure of a folding intermediate reveals the interplay between core and peripheral elements in RNA folding.

Though the molecular architecture of many native RNA structures has been characterized, the structures of folding intermediates are poorly defined. Here, we present a nucleotide-level model of a highly structured equilibrium folding intermediate of the specificity domain of the Bacillus subtilis RNase P RNA, obtained using chemical and nuclease mapping, circular dichroism spectroscopy, small-angle X-ray scattering and molecular modeling. The crystal structure indicates that the 154 nucleotide specificity domain is composed of several secondary and tertiary structural modules. The structure of the intermediate contains modules composed of secondary structures and short-range tertiary interactions, implying a sequential order of tertiary structure formation during folding. The intermediate lacks the native core and several long-range interactions among peripheral regions, such as a GAAA tetraloop and its receptor. Folding to the native structure requires the local rearrangement of a T-loop in the core in concert with the formation of the GAAA tetraloop-receptor interaction. The interplay of core and peripheral structure formation rationalizes the high degree of cooperativity observed in the folding transition leading to the native structure.

Bacillus subtilis↗
PubMed1999

Heterogeneous nuclear ribonucleoprotein I (hnRNP-I/PTB) selectively binds the conserved 3' terminus of hepatitis C viral RNA.

Hepatitis C virus (HCV) is a positive-strand RNA virus whose genome is replicated by a direct RNA-to-RNA mechanism. Initiation of negative-strand RNA synthesis is believed to proceed from the 3' end of the genomic RNA. The high conservation of the 3' terminus suggests that this region directs the assembly of proteins required for the initiation of RNA replication. We sought to determine whether host proteins bind specifically to this RNA structure. We observed specific binding of cellular proteins to labeled 3'-terminal RNA by mobility shift analysis. UV crosslinking revealed that the predominant 3'-terminal RNA-binding protein migrates as a single, 60-kDa species that can be precipitated by monoclonal antibodies directed against heterogeneous nuclear ribonucleoprotein I, also called polypyrimidine tract-binding protein (hnRNP-I/PTB), a protein previously shown to bind to the 5' internal ribosome entry site (IRES) of the HCV genome. Purified hnRNP-I/PTB also bound selectively to the 3' end of the HCV genome. hnRNP-I/PTB binding requires the upstream two stem-loop structures (SL2 and SL3) but not the most 3'-terminal stem-loop (SL1). Minor alteration of either the stem or loop sequences in SL2 or SL3 severely compromised hnRNP-I/PTB binding, suggesting extremely tight RNA structural requirements for interaction with this protein. hnRNP-I/PTB does not bind to either end of the antigenomic RNA strand and binds to the 5' IRES element of the genome at least 10-fold less avidly than to the 3' terminus. The strong, selective, and preferential binding of hnRNP-I/PTB to the 3' end of the HCV genome suggests that it may be recruited to participate in viral replication, helping to direct initiation of negative-strand RNA synthesis, stabilize the viral genome, and/or regulate encapsidation of genomic RNA.

Animals↗
PubMed1986

Secondary structure of poliovirus RNA: correlation of computer-predicted with electron microscopically observed structure.

A secondary structure map of poliovirus 1, strain Mahoney, RNA was determined by psoralen crosslinking the (+) strand and visualizing the structures in the electron microscope. Hairpins and looped hairpins were observed, and the size and distribution were measured. To orient map features the 3' end of the RNA was linked to polybromodeoxyuridine [poly(BUdR)]SV40 and histograms were constructed from these measurements. Secondary structure maps of the RNA were likewise constructed from the results of computer prediction programs for secondary structure. The programs used were those of M. Zuker (RNA2 and FOLD) which calculate a minimal global energy for a given sequence. Many single hairpins predicted by both RNA2 and FOLD showed a correlation with the histograms of hairpin structures of RNA crosslinked with psoralen. A secondary structure map was also constructed for the entire 7433 bases using the option in FOLD which allows multi-branch loops by folding uniformly stepped overlapping segments. Any structure that occurred at or greater than a given frequency was selected and mapped with respect to genome position. A correlation in structured regions was seen between psoralen-derived and computer-predicted maps of secondary structure. Furthermore, a region of large loops from base position 681 to 3899 was noted that corresponded to frequently observed large loop(s) in electron micrographs of psoralen preparations of RNA. Agreement between the two methods of determining secondary structure strengthens the credibility of the computer-aided methods used for predicting secondary structure and allows us to suggest an overall secondary structure map for poliovirus RNA.

Computers↗
PubMed1996

Magnesium ions are required by Bacillus subtilis ribonuclease P RNA for both binding and cleaving precursor tRNAAsp.

The multiple roles Mg2+ plays in ribozyme-catalyzed reactions in stabilizing RNA structure, enhancing the affinity of bound substrates, and increasing catalysis are delineated for the RNA component of ribonuclease P (RNase P RNA) by a combination of steady-state kinetics, transient kinetics, and equilibrium binding measurements. Divalent metal ions cooperatively increase the affinity of Bacillus subtilis RNase P RNA for B. subtilis tRNA(Asp) more than 10(3)-fold, consistent with at least two additional magnesium ions binding to the RNase P RNA.tRNA complex. Monovalent cations also decrease KD(tRNA) and reduce, but do not eliminate, the dependence on magnesium ions, demonstrating that nonspecific electrostatic shielding is not sufficient to explain the requirement for high salt. Both di- and monovalent cations promote the high affinity of tRNA by forming contacts in the binary complex that reduce the dissociation rate constant for tRNA. Additionally, the hyperbolic dependence of the hydrolytic rate constant on the concentration of magnesium with a K1/2 approximately equal to 36 mM suggests that a third low-affinity divalent metal ion stabilizes the transition state for pre-tRNA cleavage. Furthermore, many (about 100) magnesium ions bind independently to RNase P RNA with higher affinity than the K1/2 of any of the functionally characterized magnesium binding sites. Therefore, the magnesium binding sites that have differential affinity in either the "folded" species or binary complex are a small subset of the total number of associated magnesium ions. In summary, the importance of magnesium bound to RNase P RNA can be separated functionally into three crucial roles: at least three sites stabilize the folded RNA tertiary structure [Pan. T. (1995) Biochemistry 34, 902-909], at least two sites enhance the formation of complexes of RNase P RNA with pre-tRNA or tRNA, and at least one site stabilizes the transition state for pre-tRNA cleavage.

Bacillus subtilis↗
PubMed2005

Structure of the RNA signal essential for translational frameshifting in HIV-1.

Many pathogenic viruses use a programmed -1 translational frameshifting mechanism to regulate synthesis of their structural and enzymatic proteins. Frameshifting is vital for viral replication. A slippery sequence bound at the ribosomal A and P sites as well as a downstream stimulatory RNA structure are essential for frameshifting. Conflicting data have been reported concerning the structure of the downstream RNA signal in human immunodeficiency virus type 1 (HIV-1). Here, the solution structure of the HIV-1 frameshifting RNA signal was solved by heteronuclear NMR spectroscopy. This structure reveals a long hairpin fold with an internal three-nucleotide bulge. The internal loop introduces a bend between the lower and upper helical regions, a structural feature often seen in frameshifting pseudoknots. The NMR structure correlates with chemical probing data. The upper stem rich in conserved G-C Watson-Crick base-pairs is highly stable, whereas the bulge region and the lower stem are more flexible.

Base Composition↗
PubMed2004

Secondary structure models of the 3' untranslated regions of diverse R2 RNAs.

The RNA structure of the 3' untranslated region (UTR) of the R2 retrotransposable element is recognized by the R2-encoded reverse transcriptase in a reaction called target primed reverse transcription (TPRT). To provide insight into structure-function relationships important for TPRT, we have created alignments that reveal the secondary structure for 22 Drosophila and five silkmoth 3' UTR R2 sequences. In addition, free energy minimization has been used to predict the secondary structure for the 3' UTR R2 RNA of Forficula auricularia. The predicted structures for Bombyx mori and F. auricularia are consistent with chemical modification data obtained with beta-ethoxy-alpha-ketobutyraldehyde (kethoxal), dimethyl sulfate, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate. The structures appear to have common helices that are likely important for function.

3' Untranslated Regions↗
PubMed1994

Conformations of t-RNA: base pairing and stacking.

The Phe t-RNA structure can be fit with one point per nucleotide to lattice models, and a fit for the 76 points to a face-centered cubic lattice is achieved with an RMS of 1.76 A. There are 32 chain folds possible upon these points. Because it is impossible to calculate directly all combinations of potential base pairs for these cases, an alternative is to determine low energy secondary structures and subsequently the tertiary pairs. For each lattice fold, the low energy secondary structures are generated from a list of proximal bases. From the lists of remaining possible tertiary pairs, all combinations are generated, and these include 2,365,440 allowed conformers. Among the possible types of non-native conformational variations observed is slip pairing, accompanied by a bulge, at the end of a stem. Small changes in secondary structure can result in different tertiary pairs. Other calculations, not constrained to the t-RNA shape, are presented that involve the packing of rigid stems on a flexible internal loop. For a simple cubic lattice there are 36,484,128 lattice folds for the sixteen bases enclosing the internal loop. By attaching rigid stems and accounting for their excluded volume these are reduced to only 258,979 possible configurations. The most common stacking arrangements involve the usual two pairs of stacked stems indicated in the crystal structure. The present enumerations suggest that a completely thorough exploration of three dimensional RNA structures is feasible only with prior specification of restrictions on conformational freedom, such as those given by secondary structures.

Base Composition↗
PubMed2001

Splicing factor slt11p and its involvement in formation of U2/U6 helix II in activation of the yeast spliceosome.

Slt11p is a new splicing factor identified on the basis of synthetic lethality with a mutation in the 5' end of U2 snRNA, a region that is involved in intermolecular U2/U6 helix II interaction. Slt11p is required for spliceosome assembly. Our genetic results suggest that Slt11p is involved in the base-pairing interaction of U2/U6 helix II in vivo. We showed that the recombinant protein binds to RNAs with some degree of structural specificity. Slt11p also anneals RNA and binds to the resulting duplexes, which contain two separated helical regions. These RNA structures are reminiscent of U2/U6 helix II, which is formed concomitantly with U4/U6 stem II, and suggest that Slt11p facilitates the cooperative formation of helix II in association with stem II in the spliceosome. We show that Slt11p and Slu7p, a second-step factor, interact with each other both in vivo and in vitro and that the binding of Slu7p to Slt11p impairs the RNA-binding activity of the latter. These results suggest that the function of Slt11p is regulated by Slu7p in the spliceosome.

Amino Acid Sequence↗
PubMed2005

Molecular determinants and guided evolution of species-specific RNA editing.

Most RNA editing systems are mechanistically diverse, informationally restorative, and scattershot in eukaryotic lineages. In contrast, genetic recoding by adenosine-to-inosine RNA editing seems common in animals; usually, altering highly conserved or invariant coding positions in proteins. Here I report striking variation between species in the recoding of synaptotagmin I (sytI). Fruitflies, mosquitoes and butterflies possess shared and species-specific sytI editing sites, all within a single exon. Honeybees, beetles and roaches do not edit sytI. The editing machinery is usually directed to modify particular adenosines by information stored in intron-mediated RNA structures. Combining comparative genomics of 34 species with mutational analysis reveals that complex, multi-domain, pre-mRNA structures solely determine species-appropriate RNA editing. One of these is a previously unreported long-range pseudoknot. I show that small changes to intronic sequences, far removed from an editing site, can transfer the species specificity of editing between RNA substrates. Taken together, these data support a phylogeny of sytI gene editing spanning more than 250 million years of hexapod evolution. The results also provide models for the genesis of RNA editing sites through the stepwise addition of structural domains, or by short walks through sequence space from ancestral structures.

Adenosine↗