[Structure and function of 5S rRNA in ribosomes].
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Base Sequence↗
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Results for “RNA tertiary structure”
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The structure of an RNAi polymerase links RNA silencing and transcription.
RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-A resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world.
Amino Acid Sequence↗
Molecular architecture, structure-function relationship, and importance of the Elp3 subunit for the RNA binding of holo-elongator.
The molecular architecture of six-subunit yeast holo-Elongator complex was investigated by the use of immunoprecipitation, two-hybrid interaction mapping, and in vitro studies of binary interactions between individual subunits. Surprisingly, Elp2 is dispensable for the integrity of the holo-Elongator complex, and a purified five-subunit elp2 Delta Elongator complex retains histone acetyltransferase activity in vitro. These results indicate that the WD40 repeats in Elp2 are required neither for subunit-subunit interactions within Elongator nor for Elongator interaction with histones during catalysis. Elp2 and Elp4 were largely dispensable for the association of Elongator with nascent RNA transcript in vivo. In contrast, Elongator-RNA interaction requires the Elp3 protein. Together, these data shed light on the structure-function relationship of the Elongator complex.
Acetylation↗
Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA.
Complexes between 16S rRNA and purified ribosomal proteins, either singly or in combination, were assembled in vitro and probed with hydroxyl radicals generated from free Fe(II)-EDTA. The broad specificity of hydroxyl radicals for attack at the ribose moiety in both single- and double-stranded contexts permitted probing of nearly all of the nucleotides in the 16S rRNA chain. Specific protection of localized regions of the RNA was observed in response to assembly of most of the ribosomal proteins. The locations of the protected regions were in good general agreement with the footprints previously reported for base-specific chemical probes, and with sites of RNA-protein crosslinking. New information was obtained about interaction of ribosomal proteins with 16S rRNA, especially with helical elements of the RNA. In some cases, 5' or 3' stagger in the protection pattern on complementary strands suggests interaction of proteins with the major or minor groove, respectively, of the RNA. These results reinforce and extend previous data on the localization of ribosomal proteins with respect to structural features of 16S rRNA, and offer many new constraints for three-dimensional modeling of the 30S ribosomal subunit.
Base Sequence↗
The NMR structure of the RNA binding domain of E. coli rho factor suggests possible RNA-protein interactions.
Rho protein is an essential hexameric RNA-DNA helicase that binds nascent mRNA transcripts and terminates transcription in a wide variety of eubacterial species. The NMR solution structure of the RNA binding domain of rho, rho130, is presented. This structure consists of two sub-domains, an N-terminal three-helix bundle and a C-terminal beta-barrel that is structurally similar to the oligosaccharide/oligonucleotide binding (OB) fold. Chemical shift changes of rho130 upon RNA binding and previous mutagenetic analyses of intact rho suggest that residues Asp 60, Phe 62, Phe 64, and Arg 66 are critical for binding and support the hypothesis that ssRNA/ssDNA binding is localized in the beta-barrel sub-domain. On the basis of these studies and the tertiary structure of rho130, we propose that residues Asp 60, Phe 62, Phe 64, Arg 66, Tyr 80, Lys 105, and Arg 109 participate in RNA-protein interactions.
Binding Sites↗
Complexes of N antitermination protein of phage lambda with specific and nonspecific RNA target sites on the nascent transcript.
The mechanisms that control N protein dependent antitermination in phage lambda have counterparts in many eukaryotic systems, including specific regulatory interactions of the antitermination protein with the nascent RNA transcript. Here we describe the specific and nonspecific RNA binding modes of antitermination protein N. These modes differ markedly in RNA binding affinity and in structure. N protein, either free in solution or as a complex with nonspecific RNA, lacks observable secondary and tertiary structure and binds RNA sequences indiscriminately with a dissociation constant (Kd) of approximately 10(-6) M. In contrast N becomes partially folded with at least 16-18 amino acids of ordered alpha-helical structure and binds much more tightly (Kd approximately 10(-9) M) on forming a highly specific 1:1 complex with its cognate boxB RNA hairpin. These observations and others are used to help define a bipartite model of N-dependent antitermination in which these specific and nonspecific interactions control the binding of N to the nascent transcript. Finally the role of RNA looping in delivering the bound N to the transcription complex and determining the stability (and thus the terminator specificity) of the resulting antitermination interaction of N with the RNA polymerase is considered in quantitative terms.
Amino Acid Sequence↗
Higher order structure of chloroplastic 5S ribosomal RNA from spinach.
The secondary and tertiary structure of chloroplastic 5S ribosomal RNA from spinach was investigated by the use of several chemical and enzymatic structure probes. The four bases were monitored at one of their Watson-Crick base-pairing positions with dimethyl sulfate [at A(N1) and C(N3)] and with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate [at G(N1) and U(N3)]. Position N7 of purines was probed with diethyl pyrocarbonate (adenines) and with dimethyl sulfate (guanines). Ethylnitrosourea was used to probe phosphate involved in tertiary interaction or in cation coordination. In order to estimate the degree of stability of helices, the various chemical reagents were employed under "native" conditions (300 mM KCl and 20 mM magnesium at 37 degrees C), under "semidenaturing" conditions [1 mM ethylenediaminetetraacetic acid (EDTA) at 37 degrees C], and under denaturing conditions (1 mM EDTA at 90 degrees C). Unstructured regions were also tested with single-strand-specific nucleases T1, U2, and S1 and double-stranded or stacked regions with RNase V1 from cobra Naja naja oxiana venom. The results confirm the existence of the five helices and the two external loops proposed in the consensus model of 5S rRNA. However, the regions depicted as unpaired internal loops appear to be folded into a more complex conformation. A three-dimensional model derived from the present data and graphic modeling for a region encompassing helix IV, helix V, loop D, and loop E (nucleotides 70-110) is proposed. Nucleotides in the so-called loop E (73-79/100-106) display unusual features: Noncanonical base pairs (A-A and A-G) are formed, and three nucleotides (C75, U78, and U105) are bulging out. This region adopts an unwound and extended conformation that can be well suited for tertiary interactions or for protein binding. Several bases and phosphates candidate for the tertiary folding of the RNA were also identified.
Base Composition↗
Energetics of RNA binding by the West Nile virus RNA triphosphatase.
The West Nile virus (WNV) RNA genome harbors the characteristic methylated cap structure present at the 5' end of eukaryotic mRNAs. In the present study, we report a detailed study of the binding energetics and thermodynamic parameters involved in the interaction between RNA and the WNV RNA triphosphatase, an enzyme involved in the synthesis of the RNA cap structure. Fluorescence spectroscopy assays revealed that the initial interaction between RNA and the enzyme is characterized by a high enthalpy of association and that the minimal RNA binding site of NS3 is 13 nucleotides. In order to provide insight into the relationship between the enzyme structure and RNA binding, we also correlated the effect of RNA binding on protein structure using both circular dichroism and denaturation studies as structural indicators. Our data indicate that the protein undergoes structural modifications upon RNA binding, although the interaction does not significantly modify the stability of the protein.
Acid Anhydride Hydrolases↗
Crystal structure of a pol alpha family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9 degrees N-7.
The 2.25 A resolution crystal structure of a pol alpha family (family B) DNA polymerase from the hyperthermophilic marine archaeon Thermococcus sp. 9 degrees N-7 (9 degrees N-7 pol) provides new insight into the mechanism of pol alpha family polymerases that include essentially all of the eukaryotic replicative and viral DNA polymerases. The structure is folded into NH(2)- terminal, editing 3'-5' exonuclease, and polymerase domains that are topologically similar to the two other known pol alpha family structures (bacteriophage RB69 and the recently determined Thermococcus gorgonarius), but differ in their relative orientation and conformation. The 9 degrees N-7 polymerase domain structure is reminiscent of the "closed" conformation characteristic of ternary complexes of the pol I polymerase family obtained in the presence of their dNTP and DNA substrates. In the apo-9 degrees N-7 structure, this conformation appears to be stabilized by an ion pair. Thus far, the other apo-pol alpha structures that have been determined adopt open conformations. These results therefore suggest that the pol alpha polymerases undergo a series of conformational transitions during the catalytic cycle similar to those proposed for the pol I family. Furthermore, comparison of the orientations of the fingers and exonuclease (sub)domains relative to the palm subdomain that contains the pol active site suggests that the exonuclease domain and the fingers subdomain of the polymerase can move as a unit and may do so as part of the catalytic cycle. This provides a possible structural explanation for the interdependence of polymerization and editing exonuclease activities unique to pol alpha family polymerases. We suggest that the NH(2)-terminal domain of 9 degrees N-7 pol may be structurally related to an RNA-binding motif, which appears to be conserved among archaeal polymerases. The presence of such a putative RNA- binding domain suggests a mechanism for the observed autoregulation of bacteriophage T4 DNA polymerase synthesis by binding to its own mRNA. Furthermore, conservation of this domain could indicate that such regulation of pol expression may be a characteristic of archaea. Comparion of the 9 degrees N-7 pol structure to its mesostable homolog from bacteriophage RB69 suggests that thermostability is achieved by shortening loops, forming two disulfide bridges, and increasing electrostatic interactions at subdomain interfaces.
Amino Acid Motifs↗
Differential helix stabilities and sites pre-organized for tertiary interactions revealed by monitoring local nucleotide flexibility in the bI5 group I intron RNA.
The local environment at adenosine residues in the bI5 group I intron RNA was monitored as a function of Mg(2+) using both the traditional method of dimethyl sulfate (DMS) N1 methylation and a new approach, selective acylation of 2'-amine substituted nucleotides. These probes yield complementary structural information because N1 methylation reports accessibility at the base pairing face, whereas 2'-amine acylation scores overall residue flexibility. 2'-Amine acylation robustly detects RNA secondary structure and is sensitive to higher order interactions not monitored by DMS. Disruption of RNA structure due to the 2'-amine substitution is rare and can be compensated by stabilizing folding conditions. Peripheral helices that do not interact with other parts of the RNA are more stable than both base paired helices and tertiary interactions in the conserved catalytic core. The equilibrium state of the bI5 intron RNA, prior to assembly with its protein cofactor, thus features a relatively loosely packed core anchored by more stable external stem-loop structures. Adenosine residues in J4/5 and P9.0 form structures in which the nucleotide is constrained but the N1 position is accessible, consistent with pre-organization to form long-range interactions with the 5' and 3' splice sites.
Acylation↗
Comparative analysis of ribonuclease P RNA using gene sequences from natural microbial populations reveals tertiary structural elements.
PCR amplification of template DNAs extracted from mixed, naturally occurring microbial populations, using oligonucleotide primers complementary to highly conserved sequences, was used to obtain a large collection of diverse RNase P RNA-encoding genes. An alignment of these sequences was used in a comparative analysis of RNase P RNA secondary and tertiary structure. The new sequences confirm the secondary structure model based on sequences from cultivated organisms (with minor alterations in helices P12 and P18), providing additional support for nearly every base pair. Analysis of sequence covariation using the entire RNase P RNA data set reveals elements of tertiary structure in the RNA; the third nucleotides (underlined) of the GNRA tetraloops L14 and L18 are seen to interact with adjacent Watson-Crick base pairs in helix P8, forming A:G/C or G:A/U base triples. These experiments demonstrate one way in which the enormous diversity of natural microbial populations can be used to elucidate molecular structure through comparative analysis.
Bacteria↗
Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5'-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick.
Deinococcus radiodurans RNA ligase (DraRnl) is a template-directed ligase that seals nicked duplexes in which the 3'-OH strand is RNA. DraRnl is a 342 amino acid polypeptide composed of a C-terminal adenylyltransferase domain fused to a distinctive 126 amino acid N-terminal module (a putative OB-fold). An alanine scan of the C domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs I, Ia, III, IIIa, IV and V. Seven mutants were dysfunctional by virtue of defects in ligase adenylylation: T163A, H167A, G168A, K186A, E230A, F281A and E305A. Four of these were also defective in phosphodiester formation at a preadenylylated nick: G168A, E230A, F281A and E305A. Two nick sealing-defective mutants were active in ligase adenylylation and sealing a preadenylylated nick, thereby implicating Ser185 and Lys326 in transfer of AMP from the enzyme to the nick 5'-PO(4). Whereas deletion of the N-terminal domain suppressed overall nick ligation and ligase adenylylation, it did not compromise sealing at a preadenylylated nick. Mutational analysis of 15 residues of the N domain identified Lys26, Gln31 and Arg79 as key constituents. Structure-activity relationships at the essential residues were determined via conservative substitutions. We propose that DraRnl typifies a new clade of polynucleotide ligases. DraRnl homologs are detected in several eukaryal proteomes.
Alanine↗
The NMR structure of Escherichia coli ribosomal protein L25 shows homology to general stress proteins and glutaminyl-tRNA synthetases.
The structure of the Escherichia coli ribosomal protein L25 has been determined to an r.m.s. displacement of backbone heavy atoms of 0.62 +/- 0.14 A by multi-dimensional heteronuclear NMR spectroscopy on protein samples uniformly labeled with 15N or 15N/13C. L25 shows a new topology for RNA-binding proteins consisting of a six-stranded beta-barrel and two alpha-helices. A putative RNA-binding surface for L25 has been obtained by comparison of backbone 15N chemical shifts for L25 with and without a bound cognate RNA containing the eubacterial E-loop that is the site for binding of L25 to 5S ribosomal RNA. Sequence comparisons with related proteins, including the general stress protein, CTC, show that the residues involved in RNA binding are highly conserved, thereby providing further confirmation of the binding surface. Tertiary structure comparisons indicate that the six-stranded beta-barrels of L25 and of the tRNA anticodon-binding domain of glutaminyl-tRNA synthetase are similar.
Amino Acid Sequence↗
Molecular mapping of the conductance activity linked to tAE1 expressed in Xenopus oocyte.
It was previously shown that expressed in Xenopus oocyte the trout (tAE1) and the mouse (mAE1) anion exchangers behave differently: both elicit anion exchange activity but only tAE1 induces a transport of organic solutes correlated with an anion conductance. In order to identify the structural domains involved in the induction of tAE1 channel activity, chimeras have been prepared between mouse and trout AE1. As some constructs were not expressed at the plasma membrane, skate exchanger (skAE1) was used instead of mouse exchanger to complete the structure-function analysis. The present paper shows that skAE1, highly similar to mAE1, does not induce a chloride conductance when expressed in Xenopus oocyte. Construct expression analysis showed that only tAE1 transmembrane domain is linked to the anion conductance. More precisely, we identified two regions composed of helices 6, 7 and 8 and putative helices 12 and 13 which are required for this function.
Animals↗
Crystal structure of Thermus aquaticus Gfh1, a Gre-factor paralog that inhibits rather than stimulates transcript cleavage.
Transcription elongation in bacteria is promoted by Gre-factors, which stimulate an endogenous, endonucleolytic transcript cleavage activity of the RNA polymerase. A GreA paralog, Gfh1, present in Thermus aquaticus and Thermus thermophilus, has the opposite effect on elongation complexes, inhibiting rather than stimulating transcript cleavage. We have determined the 3.3 angstroms-resolution X-ray crystal structure of T.aquaticus Gfh1. The structure reveals an N-terminal and a C-terminal domain with close structural similarity to the domains of GreA, but with an unexpected conformational change in terms of the orientation of the domains with respect to each other. However, structural and functional analysis suggests that when complexed with RNA polymerase, Gfh1 adopts a conformation similar to that of GreA. These results reveal considerable structural flexibility for Gfh1, and for Gre-factors in general, as suggested by structural modeling, and point to a possible role for the conformational switch in Gre-factor and Gfh1 regulation. The opposite functional effect of Gfh1 compared with GreA may be determined by three structural characteristics. First, Gfh1 lacks the basic patch present in Gre-factors that likely plays a role in anchoring the 3'-fragment of the back-tracked RNA. Second, the loop at the tip of the N-terminal coiled-coil is highly flexible and contains extra acidic residues compared with GreA. Third, the N-terminal coiled-coil finger lacks a kink in the first alpha-helix, resulting in a straight coiled-coil compared with GreA. The latter two characteristics suggest that Gfh1 chelates a magnesium ion in the RNA polymerase active site (like GreA) but in a catalytically inactive configuration.
Amino Acid Sequence↗
Structural basis for substrate recognition by the editing domain of isoleucyl-tRNA synthetase.
In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNA(Ile), in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 A resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120 degrees and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180 degrees . The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T.thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.
Amino Acid Sequence↗
Distinct domains mediate the early and late functions of the Drosophila ovarian tumor proteins.
The Drosophila melanogaster ovarian tumor (otu) gene encodes two novel protein isoforms that are required at multiple stages of oogenesis. We have examined the activity of a set of C-terminal truncation Otu proteins as well as a GFP-tagged Otu (Otu-GFP). These experiments have shown that a putative Tudor domain in the central region of the large Otu isoform and a separate domain in the C-terminal region are required for regulation of cyst formation and oocyte maturation, respectively. We also present evidence that a portion of Otu co-fractionates with mRNA/protein complexes (mRNPs) and show that Otu-GFP associates with cytoplasmic aggregates at periphery of the nucleus at an intermediate stage of oogenesis. This study substantially clarifies the relationship between Otu structure and function and reveals new clues about interacting components.
Animals↗
Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A:eEF1Balpha.
The crystal structure of a complex between the protein biosynthesis elongation factor eEF1A (formerly EF-1alpha) and the catalytic C terminus of its exchange factor, eEF1Balpha (formerly EF-1beta), was determined to 1.67 A resolution. One end of the nucleotide exchange factor is buried between the switch 1 and 2 regions of eEF1A and destroys the binding site for the Mg(2+) ion associated with the nucleotide. The second end of eEF1Balpha interacts with domain 2 of eEF1A in the region hypothesized to be involved in the binding of the CCA-aminoacyl end of the tRNA. The competition between eEF1Balpha and aminoacylated tRNA may be a central element in channeling the reactants in eukaryotic protein synthesis. The recognition of eEF1A by eEF1Balpha is very different from that observed in the prokaryotic EF-Tu:EF-Ts complex. Recognition of the switch 2 region in nucleotide exchange is, however, common to the elongation factor complexes and those of Ras:Sos and Arf1:Sec7.
Binding Sites↗