RNAPII: a specific target for the cell cycle kinase complex.
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Cell Cycle↗
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Results for “RNA tertiary structure”
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Tertiary structure determinants in transfer RNA. I. Pseudouridine.
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Alkylation↗
Yeast tRNA(Met) recognition by methionyl-tRNA synthetase requires determinants from the primary, secondary and tertiary structure: a review.
The primordial role of the CAU anticodon in methionine identity of the tRNA has been established by others nearly a decade ago in Escherichia coli and yeast tRNA(Met). We show here that the CAU triplet alone is unable to confer methionine acceptance to a tRNA. This requires the contribution of the discriminatory base A73 and the non-anticodon bases of the anticodon loop. To better understand the functional communication between the anticodon and the active site, we analysed the binding and aminoacylation of tRNA(Met) based anticodon and acceptor-stem minihelices and of tRNA(Met) chimeras where the central core region of yeast tRNA(Met) is replaced by that of unusual mitochondrial forms lacking either a D-stem or a T-stem. These studies suggest that the high selectivity of the anticodon bases in tRNA(Met) implies the L-conformation of the tRNA and the presence of a D-stem. The importance of a L-structure for recognition of tRNA(Met) was also deduced from mutations of tertiary interactions known to play a general role in tRNA(Met) folding.
Amino Acid Sequence↗
NMR structure of varkud satellite ribozyme stem-loop V in the presence of magnesium ions and localization of metal-binding sites.
In the Neurospora VS ribozyme, magnesium ions facilitate formation of a loop-loop interaction between stem-loops I and V, which is important for recognition and activation of the stem-loop I substrate. Here, we present the high-resolution NMR structure of stem-loop V (SL5) in the presence of Mg(2+) (SL5(Mg)) and demonstrate that Mg(2+) induces a conformational change in which the SL5 loop adopts a compact structure with most characteristics of canonical U-turn structures. Divalent cation-binding sites were probed with Mn(2+)-induced paramagnetic line broadening and intermolecular NOEs to Co(NH(3))(6)(3+). Structural modeling of Mn(H(2)O)(6)(2+) in SL5(Mg) revealed four divalent cation-binding sites in the loop. Sites 1, 3, and 4 are located in the major groove near multiple phosphate groups, whereas site 2 is adjacent to N7 of G697 and N7 of A698 in the minor groove. Cation-binding sites equivalent to sites 1-3 in SL5 are present in other U-turn motifs, and these metal-binding sites may represent a common feature of the U-turn fold. Although magnesium ions affect the loop conformation, they do not significantly change the conformation of residues 697-699 involved in the proposed Watson-Crick base pairs with stem-loop I. In both the presence and the absence of Mg(2+), G697, A698, and C699 adopt an A-form structure that exposes their Watson-Crick faces, and this is compatible with their proposed interaction with stem-loop I. In SL5(Mg), however, U700 becomes exposed on the minor groove face of the loop in the proximity of the bases of G697, A698, and C699, suggesting that the Mg(2+)-bound conformation of stem-loop V allows additional contacts with stem-loop I. These studies improve our understanding of the role of Mg(2+) in U-turn structures and in substrate recognition by the VS ribozyme.
Base Pairing↗
Structural basis for anticodon recognition by discriminating glutamyl-tRNA synthetase.
Glutamyl-tRNA synthetases (GluRSs) are divided into two distinct types, with regard to the presence or absence of glutaminyl-tRNA synthetase (GlnRS) in the genetic translation systems. In the original 19-synthetase systems lacking GlnRS, the 'non-discriminating' GluRS glutamylates both tRNAGlu and tRNAGln. In contrast, in the evolved 20-synthetase systems with GlnRS, the 'discriminating' GluRS aminoacylates only tRNAGlu. Here we report the 2.4 A resolution crystal structure of a 'discriminating' GluRS.tRNAGlu complex from Thermus thermophilus. The GluRS recognizes the tRNAGlu anticodon bases via two alpha-helical domains, maintaining the base stacking. We show that the discrimination between the Glu and Gln anticodons (34YUC36 and 34YUG36, respectively) is achieved by a single arginine residue (Arg 358). The mutation of Arg 358 to Gln resulted in a GluRS that does not discriminate between the Glu and Gln anticodons. This change mimics the reverse course of GluRS evolution from anticodon 'non-dicsriminating' to 'discriminating'.
Anticodon↗
Novel mutations of the transglutaminase 1 gene in lamellar ichthyosis.
Lamellar ichthyosis, one form of congenital autosomal recessive ichthyosis, is caused by mutations in the gene (TGM1) encoding the transglutaminase 1 enzyme. Mutations, deletions, or insertion of TGM1 have been reported so far. Here we report that three novel mutations of TGM1, D101V, N288T, and R306W, cause lamellar ichthyosis in two different families. The patient in family LI-KD has N288T and R306W mutations, and the patient in family LI-LK has D101V and R306W mutations. The activity of the transglutaminase 1 enzyme of the patient in family LI-LK was only about 15% of normal. Also, three-dimensional structural prediction analyses revealed that the N288T and R306W mutations, and possibly the D101V mutation, cause misfolding in the central catalytic core domain of the transglutaminase 1 enzyme that would probably result in reduced enzyme activity. Our data suggest that the greatly reduced transglutaminase 1 activities are due to disruptions of the native folding of transglutaminase 1, and that these mutations may play a critical role in the pathology of lamellar ichthyosis.
Amino Acid Substitution↗
Characterization of a low redox potential laccase from the basidiomycete C30.
A new exocellular laccase was purified from the basidiomycete C30. LAC2 is an acidic protein (pI = 3.2) preferentially produced upon a combined induction by copper and p-hydroxybenzoate. The spectroscopic signature (UV/visible and EPR) of this isoform is typical of multicopper oxidases, but its enzymatic and physico-chemical properties proved to be markedly different from those of LAC1, the constitutive laccase previously purified from the same organism. In particular, the LAC2 kcat values observed for the oxidation of the substrates syringaldazine (kcat = 65 600 min-1), ABTS (2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (kcat = 41 000 min-1) and guaiacol (kcat = 75 680 min-1) are 10-40 times those obtained with LAC1 and the redox potential of its T1 copper is 0.17 V lower than that of LAC1 (E degrees = 0.73 V). This is the first report on a single organism producing simultaneously both a high and a low redox potential laccase. The cDNA, clac2, was cloned and sequenced. It encodes a protein of 528 amino acids that shares 69% identity (79% similarity) with LAC1 and 81% identity (95% similarity) with Lcc3-2 from Polyporus ciliatus (AF176321-1), its nearest neighbor in database. Possible reasons for why this basidiomycete produces, in vivo, enzyme forms with such different behaviors are discussed.
Azides↗
Functional communication in the recognition of tRNA by Escherichia coli glutaminyl-tRNA synthetase.
Wild-type Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) poorly aminoacylates opal suppressors (GLN) derived from tRNA(Gln). Mutations in glnS (the gene encoding GlnRS) that compensate for impaired aminoacylation were isolated by genetic selection. Two glnS mutants were obtained by using opal suppressors differing in the nucleotides composing the base pair at 3.70: glnS113 with an Asp-235-->Asn change selected with GLNA3U70 (GLN carrying G3-->A and C70-->U changes), and glnS114 with a Gln-318-->Arg change selected with GLNU70 (GLN carrying a C70-->U change). The Asp-235-->Asn change was identified previously by genetic selection. Additional mutants were isolated by site-directed mutagenesis followed by genetic selection; the mutant enzymes have single amino acid changes (Lys-317-->Arg and Gln-318-->Lys). A number of mutants with no phenotype also were obtained randomly. In vitro aminoacylation of a tRNA(Gln) transcript by GlnRS enzymes with Lys-317-->Arg, Gln-318-->Lys, or Gln-318-->Arg changes shows that the enzyme's kinetic parameters are not greatly affected by the mutations. However, aminoacylation of a tRNA(Gln) transcript with an opal (UCA) anticodon shows that the specificity constants (kcat/Km) for the mutant enzymes were 5-10 times above that of the wild-type GlnRS. Interactions between Lys-317 and Gln-318 with the inside of the L-shaped tRNA and with the side chain of Gln-234 provide a connection between the acceptor end-binding and anticodon-binding domains of GlnRS. The GlnRS mutants isolated suggest that perturbation of the interactions with the inside of the tRNA L shape results in relaxed anticodon recognition.
Amino Acid Sequence↗
On the mechanism of action of 9-O-arylalkyloxime derivatives of 6-O-mycaminosyltylonolide, a new class of 16-membered macrolide antibiotics.
New 16-membered 9-aryl-alkyl oxime derivatives of 5-O-mycaminosyl-tylonolid (OMT) have recently been prepared and were found to exhibit high activity against macrolide-resistant strains. In this study, we show that these compounds do not affect the binding of tRNAs to ribosomes in a cell-free system derived from Escherichia coli and that they cannot inhibit peptidyltransferase, peptidyl-tRNA translocation, or poly(U)-dependent poly(Phe) synthesis. However, they severely inhibit poly(A)-dependent poly(Lys) synthesis and compete with erythromycin or tylosin for binding to common or partially overlapping sites in the ribosome. According to footprinting analysis, the lactone ring of these compounds seems to occupy the classic binding site of macrolides that is located at the entrance of the exit tunnel, whereas the extending alkyl-aryl side chain seems to penetrate deeper in the tunnel, where it protects nucleoside A752 in domain II of 23S rRNA. In addition, this side chain causes an increased affinity for mutant ribosomes that may be responsible for their effectiveness against macrolide resistant strains. As revealed by detailed kinetic analysis, these compounds behave as slow-binding ligands interacting with functional ribosomal complexes through a one-step mechanism. This type of inhibitor has several attractive features and offers many chances in designing new potent drugs.
Anti-Bacterial Agents↗
Structural analysis of human growth hormone with respect to the dominant expression of growth hormone (GH) mutations in isolated GH deficiency type II.
Human GH protein consists of four alpha-helices and contains two disulfide bridges. Isolated GH deficiency type II (IGHD II) is mainly caused by heterozygous splice site mutations of GH-1 leading to the disruption of one disulfide bridge (Cys53-Cys165) and to the loss of amino acids (aa) 32-71, which comprise the complete loop between alpha-helices 1 and 2. The mutant GH protein exerts a dominant negative effect on wild-type (wt) GH secretion by unclear mechanisms. For study of the structure-function relationship of GH mutants concerning the dominant negative effect, expression vectors harboring mutated GH cDNAs were transiently cotransfected with a vector encoding wtGH (pwtGH) into GH4C1 cells. Plasmids encoding beta-galactosidase, luciferase, or IGF-binding protein-2 were cotransfected with pwtGH and either of the GH mutants. Compared with the control transfection with pwtGH, GH secretion was mildly decreased by coexpressing wtGH and different GH point mutants with isolated disruption of the disulfide bridge Cys53-Cys165. Similar results were observed with GH mutants deleted in aa 32-46 or 32-52. Deletion of more aa (32-53, 32-63, 32-69, 32-71) ascendingly decreased GH secretion and content in parallel with the increasing length of the deleted stretch. An inhibitory dose-dependent effect of del32-69GH and del32-71GH on the activity/amount of coexpressed beta-galactosidase, luciferase, and IGF-binding protein-2 was found, whereas mRNA levels were unaffected. Hence, the extent of deletion played the major role in expression of the dominant negative effect. The inhibitory effect of GH mutants on heterologously expressed, non-GH proteins suggests that the dominant negative effect is not limited to GH or to proteins of the regulated secretory pathway, but may depend on expression levels.
Binding Sites↗
Cysteine repeat domains and adjacent sequences determine distinct bone morphogenetic protein modulatory activities of the Drosophila Sog protein.
The Drosophila short gastrulation gene (sog) encodes a large extracellular protein (Sog) that inhibits signaling by BMP-related ligands. Sog and its vertebrate counterpart Chordin contain four copies of a cysteine repeat (CR) motif defined by 10 cysteine residues spaced in a fixed pattern and a tryptophan residue situated between the first two cysteines. Here we present a structure-function analysis of the CR repeats in Sog, using a series of deletion and point mutation constructs, as well as constructs in which CR domains have been swapped. This analysis indicates that the CR domains are individually dispensable for Sog function but that they are not interchangeable. These studies reveal three different types of Sog activity: intact Sog, which inhibits signaling mediated by the ligand Glass bottom boat (Gbb), a more broadly active class of BMP antagonist referred to as Supersog, and a newly identified activity, which may promote rather than inhibit BMP signaling. Analysis of the activities of CR swap constructs indicates that the CR domains are required for full activity of the various forms of Sog but that the type of Sog activity is determined primarily by surrounding protein sequences. Cumulatively, our analysis suggests that CR domains interact physically with adjacent protein sequences to create forms of Sog with distinct BMP modulatory activities.
Alanine↗
Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer.
The complex formed by the hairpin ribozyme and its substrate consists of two independently folding domains which interact to form a catalytic structure. Fluorescence resonance energy transfer methods permit us to study reversible transitions of the complex between open and closed forms. Results indicate that docking of the domains is required for both the cleavage and ligation reactions. Docking is rate-limiting for ligation (2 min-1) but not for cleavage, where docking (0.5 min-1) precedes a rate-limiting conformational transition or slow-reaction chemistry. Strikingly, most modifications to the RNA (such as a G+1A mutation in the substrate) or reaction conditions (such as omission of divalent metal ion cofactors) which inhibit catalysis do so by preventing docking. This demonstrates directly that mutations and modifications which inhibit a step following substrate binding are not necessarily involved in catalysis. An improved kinetic description of the catalytic cycle is derived, including specific structural transitions.
Base Composition↗
Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization.
Retroviruses selectively package two copies of their RNA genomes in the context of a large excess of nongenomic RNA. Specific packaging of genomic RNA is achieved, in part, by recognizing RNAs that form a poorly understood dimeric structure at their 5' ends. We identify, quantify the stability of, and use extensive experimental constraints to calculate a 3D model for a tertiary structure domain that mediates specific interactions between RNA genomes in a gamma retrovirus. In an initial interaction, two stem-loop structures from one RNA form highly stringent cross-strand loop-loop base pairs with the same structures on a second genomic RNA. Upon subsequent folding to the final dimer state, these intergenomic RNA interactions convert to a high affinity and compact tertiary structure, stabilized by interdigitated interactions between U-shaped RNA units. This retroviral conformational switch model illustrates how two-step formation of an RNA tertiary structure yields a stringent molecular recognition event at early assembly steps that can be converted to the stable RNA architecture likely packaged into nascent virions.
Base Sequence↗
Structural bases of the annealing of primer tRNA(3Lys) to the HIV-1 viral RNA.
To promote the initiation of reverse transcription, the HIV-1 virus uses a host tRNA as a primer, tRNA(3Lys). The annealing of tRNA(3Lys) to the viral RNA requires the breaking of the 3D structure of the tRNA and RNA rearrangements, to form a stable initiation complex recognised by the reverse transcriptase. The annealing is mediated by a viral factor, the nucleocapsid protein. This protein has been studied for a long while to define the role of its different sub-domains and their mode of action. Only recently, a consensus view seems to emerge. The structure of the initiation complex, is still discussed. The goal of this review is to clarify what is known about the formation and the structure of the tRNA/viral RNA complex and the role of the nucleocapsid protein from a structural point of view.
Base Sequence↗
Bases defining an ammonium and magnesium ion-dependent tertiary structure within the large subunit ribosomal RNA.
A 58 nucleotide RNA derived from a highly conserved domain of the large subunit ribosomal RNA (Escherichia coli 1051 to 1108) has a set of tertiary interactions that is stabilized by NH4+ and Mg2+ in preference to other ions. We have mapped the nucleotides contributing to this structure by examining the thermal denaturation of 25 sequence variants. Where necessary compensatory mutations were made to preserve the phylogenetically conserved secondary structure. Substitutions of bases or base-pairs at eight positions specifically eliminate the ion-dependent tertiary structure without affecting the secondary structure stability; most of these positions are conserved among all large subunit RNA sequences. At two positions, substitutions of bases found in other organisms stabilize the E. coli tertiary structure by substantial amounts (delta G 37 degrees becomes more favorable by -1.8 to -4.5 kcal/mol). One of these variants disrupts a potential A.U base-pair within a helix, suggesting that the tertiary structure competes with alternative structures. The results show that this rRNA domain contains an extensive, highly conserved, and very stable set of tertiary interactions. The sequence in E. coli, and probably most other organisms, has not evolved to maximum stability. It is possible that natural selection has "tuned" the tertiary structure to an optimum stability, perhaps because the structure must open and close during the ribosome cycle.
Base Composition↗
Protein facilitation of group I intron splicing by assembly of the catalytic core and the 5' splice site domain.
The yeast mitochondrial group I intron b15 undergoes self-splicing at high Mg2+ concentrations, but requires the splicing factor CBP2 for reaction under physiological conditions. Chemical accessibility and UV cross-linking experiments now reveal that self-processing is slow because functional elements are not properly positioned in an active tertiary structure. Folding energy provided by CBP2 drives assembly of two RNA domains that comprise the catalytic core and meditates association of an approximately 100 nt 5' domain that contains the 5' splice site. Thus, the protein assembles RNA secondary structure elements into a specific three-dimensional array while the RNA provides the catalytic center. The division of labor between RNA and protein illustrated by this simple system reveals principles applicable to complex ribonucleoprotein assemblies such as the spliceosome and ribosome.
Base Sequence↗
Quantitative determination of the number of secondary and tertiary structure base pairs in transfer RNA in solution.
Resonances in the low-field (11-15 ppm) nuclear magnetic resonance spectrum (NMR) of tRNA molecules arise from secondary and tertiary structure base pairs (1 resonance for each base pair) as well as tertiary structure hydrogen bonds. An accurate method for integrating the low-field spectra has been developed and applied to seven different tRNA. In the presence of high levels of magnesium (10 mM free magnesium) the number of resonances (base pairs) per molecule is typically 3-4 more than the number predicted by the cloverleaf model. These results confirm our recent proposal that, under proper conditions, most tRNA exhibit 3-4 tertiary structure interactions in solution, which are also observed in x-ray diffraction studies of yeast tRNAPhe. In addition to common resonances in the 11-15 ppm region, there are common resonances at 10.5 and 9.5 ppm. A critique of methods used to integrate the low-field spectra is given and possible sources of error are indicated. The discrepancy between our present results and previous studies, which indicated that the number of base pairs per molecule was close to the number predicted by the cloverleaf model, can be attributed partly to differences in magnesium concentration and partly to inaccuracies inherent in the integration methods used.
Escherichia coli↗
RNA synthesis in a cage--structural studies of reovirus polymerase lambda3.
The reovirus polymerase and those of other dsRNA viruses function within the confines of a protein capsid to transcribe the tightly packed dsRNA genome segments. The crystal structure of the reovirus polymerase, lambda3, determined at 2.5 A resolution, shows a fingers-palm-thumb core, similar to those of other viral polymerases, surrounded by major N- and C-terminal elaborations, which create a cage-like structure, with four channels leading to the catalytic site. This "caged" polymerase has allowed us to visualize the results of several rounds of RNA polymerization directly in the crystals. A 5' cap binding site on the surface of lambda3 suggests a template retention mechanism by which attachment of the 5' end of the plus-sense strand facilitates insertion of the 3' end of the minus-sense strand into the template channel.
Amino Acid Sequence↗