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1.
Crystal structure of human Pus10, a novel pseudouridine synthase 总被引:2,自引:0,他引:2
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Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA
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Massenet S Motorin Y Lafontaine DL Hurt EC Grosjean H Branlant C 《Molecular and cellular biology》1999,19(3):2142-2154
Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism. 相似文献
4.
RNA-guided pseudouridine (Psi) synthesis in Archaea and Eukarya requires a four-protein one-RNA containing box H/ACA ribonucleoprotein (RNP) complex. The proteins in the archaeal RNP are aCbf5, aNop10, aGar1 and L7Ae. Pyrococcus aCbf5-aNop10 is suggested to be the minimal catalytic core in this synthesis and the activity is enhanced by L7Ae and aGar1. The protein aCbf5 is homologous to eukaryal Cbf5 (dyskerin, NAP57) as well as to bacterial TruB and eukaryal Pus4; the last two produce YPsi55 in tRNAs in a guide RNA-independent manner. Here, using recombinant Methanocaldococcus jannaschii proteins, we report that aCbf5 and aGar1 together can function as a tRNA Psi55 synthase in a guide RNA-independent manner. This activity is enhanced by aNop10, but not by L7Ae. The aCbf5 alone can also produce Psi55 in tRNAs that contain the canonical 3'-CCA sequence and this activity is stimulated by aGar1. These results suggest that the roles of accessory proteins are different in guide RNA-dependent and independent Psi synthesis by aCbf5. The presence of conserved C (or U) and A at tRNA positions 56 and 58, respectively, which are required for TruB/Pus4 activity, is not essential for aCbf5-mediated Psi55 formation. Conserved A58 in tRNA normally forms a tertiary reverse Hoogstein base pair with an equally conserved U54. This base pair is recognized by TruB. Apparently aCbf5 does not require this base pair to recognize U55 for conversion to Psi55. 相似文献
5.
Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.
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The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle. 相似文献
6.
We describe the first identification of pseudouridine (Psi) residues in ribosomal RNA (23S rRNA) of an hyperthermophilic Archaea Sulfolobus acidocaldarius. In contrast to Eucarya rRNA, only six Psi residues were detected, which is rather close to the situation in Bacteria. However, three modified positions (Psi(2479), Psi(2535) and Psi(2550)) are unique for S. acidocaldarius. Two Psi residues at positions 2060 and 2594 are universally conserved, while one other Psi (position 2066) is also common to Eucarya. Taken together the results argue against the conservation of Psi-synthases between Archaea and Bacteria and provide a basis for the search of snoRNA-like guides for Psi formation in Archaea. 相似文献
7.
Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two in the Y16S mutant and one in the Y16F and FFF mutants, with intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of (1)H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less probable in WT KSI. 相似文献
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Ribosome structure and activity are altered in cells lacking snoRNPs that form pseudouridines in the peptidyl transferase center 总被引:1,自引:0,他引:1
One of the oldest questions in RNA science is the role of nucleotide modification. Here, the importance of pseudouridine formation (Psi) in the peptidyl transferase center of rRNA was examined by depleting yeast cells of 1-5 snoRNAs that guide a total of six Psi modifications. Translation was impaired substantially with loss of a conserved Psi in the A site of tRNA binding. Depletion of other Psis had subtle or no apparent effect on activity; however, synergistic effects were observed in some combinations. Pseudouridines are proposed to enhance ribosome activity by altering rRNA folding and interactions, with some Psis having greater effects than others. The possibility that modifying snoRNPs might affect ribosome structure in other ways is also discussed. 相似文献
10.
The pseudouridine synthase RluD is required for normal ribosome assembly and function in Escherichia coli
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RluD is the pseudouridine synthase responsible for the formation of Psi1911, Psi1915, and Psi1917 in Escherichia coli 23S rRNA. Previous work from our laboratory demonstrated that disruption of the rluD gene and/or loss of the pseudouridine residues for which it is responsible resulted in a severe growth phenotype. In the current work we have examined further the effect of the loss of the RluD protein and its product pseudouridine residues in a deletion strain lacking the rluD gene. This strain exhibits defects in ribosome assembly, biogenesis, and function. Specifically, there is a deficit of 70S ribosomes, an increase in 50S and 30S subunits, and the appearance of new 62S and 39S particles. Analysis of the 39S particles indicates that they are immature precursors of the 50S subunits, whereas the 62S particles are derived from the breakdown of unstable 70S ribosomes. In addition, purified mutant 70S ribosomes were found to be somewhat less efficient than wild type in protein synthesis. The defect in ribosome assembly and resulting growth phenotype of the mutant could be restored by expression of wild-type RluD and synthesis of Psi1911, Psi1915, and Psi1917 residues, but not by catalytically inactive mutant RluD proteins, incapable of pseudouridine formation. The data suggest that the loss of the pseudouridine residues can account for all aspects of the mutant phenotype; however, a possible second function of the RluD synthase is also discussed. 相似文献
11.
Crystal structure of the highly divergent pseudouridine synthase TruD reveals a circular permutation of a conserved fold 总被引:2,自引:0,他引:2
The pseudouridine (Psi) synthases Pus7p and TruD define a family of RNA-modifying enzymes with no sequence similarity to previously characterized Psi synthases. The 2.2 A resolution structure of Escherichia coli TruD reveals a U-shaped molecule with a catalytic domain that superimposes closely on that of other Psi synthases. A domain that appears to be unique to TruD/Pus7p family enzymes hinges over the catalytic domain, possibly serving to clasp the substrate RNAs. The active site comprises residues that are conserved in other Psi synthases, although at least one comes from a structurally distinct part of the protein. Remarkably, the connectivity of the structural elements of the TruD catalytic domain is a circular permutation of that of its paralogs. Because the sequence of the permuted segment, a beta-strand that bisects the catalytic domain, is conserved among orthologs from bacteria, archaea and eukarya, the permutation likely happened early in evolution. 相似文献
12.
Nadine Czudnochowski Gary W. Ashley Daniel V. Santi Akram Alian Janet Finer-Moore Robert M. Stroud 《Nucleic acids research》2014,42(3):2037-2048
RluB catalyses the modification of U2605 to pseudouridine (Ψ) in a stem-loop at the peptidyl transferase center of Escherichia coli 23S rRNA. The homolog RluF is specific to the adjacent nucleotide in the stem, U2604. The 1.3 Å resolution crystal structure of the complex between the catalytic domain of RluB and the isolated substrate stem-loop, in which the target uridine is substituted by 5-fluorouridine (5-FU), reveals a covalent bond between the isomerized target base and tyrosine 140. The structure is compared with the catalytic domain alone determined at 2.5 Å resolution. The RluB-bound stem-loop has essentially the same secondary structure as in the ribosome, with a bulge at A2602, but with 5-FU2605 flipped into the active site. We showed earlier that RluF induced a frame-shift of the RNA, moving A2602 into the stem and translating its target, U2604, into the active site. A hydrogen-bonding network stabilizes the bulge in the RluB–RNA but is not conserved in RluF and so RluF cannot stabilize the bulge. On the basis of the covalent bond between enzyme and isomerized 5-FU we propose a Michael addition mechanism for pseudouridine formation that is consistent with all experimental data. 相似文献
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Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site. 相似文献
14.
Crystal structure of the catalytic domain of RluD, the only rRNA pseudouridine synthase required for normal growth of Escherichia coli
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Escherichia coli pseudouridine synthase RluD makes pseudouridines 1911, 1915, and 1917 in the loop of helix 69 in 23S RNA. These are the most highly conserved ribosomal pseudouridines known. Of 11 pseudouridine synthases in E. coli, only cells lacking RluD have severe growth defects and abnormal ribosomes. We have determined the 2.0 A structure of the catalytic domain of RluD (residues 77-326), the first structure of an RluA family member. The catalytic domain folds into a mainly antiparallel beta-sheet flanked by several loops and helices. A positively charged cleft that presumably binds RNA leads to the conserved Asp 139. The RluD N-terminal S4 domain, connected by a flexible linker, is disordered in our structure. RluD is very similar in both catalytic domain structure and active site arrangement to the pseudouridine synthases RsuA, TruB, and TruA. We identify five sequence motifs, two of which are novel, in the RluA, RsuA, TruB, and TruA families, uniting them as one superfamily. These results strongly suggest that four of the five families of pseudouridine synthases arose by divergent evolution. The RluD structure also provides insight into its multisite specificity. 相似文献
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Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) plays a central metabolic role in photosynthetic eukaryotes, and its catabolism is a crucial process for the nutrient economy of higher plants. The rubisco holoenzyme is assembled from eight chloroplast-encoded large subunits and eight nuclear-encoded small subunits. We have identified a cluster of conserved tyrosines at the interface between subunits (comprising Y67, Y68, and Y72 from the betaA-betaB loop of the small subunit and Y226 from the large subunit) that may contribute to holoenzyme stability. To investigate the role of these tyrosines in rubisco structure and in vivo degradation, we have examined site-directed mutants of these residues (Y67A, Y68A, Y72A, and Y226L) in Chlamydomonas reinhardtii. Even if all mutant strains were able to grow photoautotrophically, they exhibited a reduction in rubisco activity and/or the level of expression, especially the Y67A and Y72A mutants. Besides, all mutant rubiscos were inactivated at a lower temperature than the wild type. The kinetics of proteolysis of the mutant enzymes with subtilisin revealed structural alterations, leading to facilitated disassembly (in the cases of Y67A and Y72A) or aggregation propensity (for Y68A and Y226L). When subjected to oxidative stress in vivo through exposure of liquid cultures to hydrogen peroxide, all mutant strains degraded rubisco at a faster rate than the wild type. These results demonstrate that the tyrosine cluster around the betaA-betaB loop of rubisco small subunit plays a stabilizing role by affecting the catalytic activity and the degradation rate of the enzyme in stressed cells. 相似文献
16.
Gustafson CL Stauffacher CV Hallenga K Van Etten RL 《Protein science : a publication of the Protein Society》2005,14(10):2515-2525
Eukaryotic low-molecular-weight protein tyrosine phosphatases (LMW PTPs) contain a conserved serine, a histidine with an elevated pKa, and an active site asparagine that together form a highly conserved hydrogen bonding network. This network stabilizes the active site phosphate binding loop for optimal substrate binding and catalysis. In the phosphatase from the bovine parasite Tritrichomonas foetus (TPTP), both the conserved serine (S37) and asparagine (N14) are present, but the conserved histidine has been replaced by a glutamine residue (Q67). Site-directed mutagenesis, kinetic, and spectroscopic experiments suggest that Q67 is located near the active site and is important for optimal catalytic activity. Kinetic experiments also suggest that S37 participates in the active site/hydrogen bonding network. Nuclear magnetic resonance spectroscopy was used to determine the three-dimensional structure of the TPTP enzyme and to further examine the roles of S37 and Q67. The backbone conformation of the TPTP phosphate binding loop is nearly superimposable with that of other tyrosine phosphatases, with N14 existing in a strained, left-handed conformation that is a hallmark of the active site hydrogen bonding network in the LMW PTPs. As expected, both S37 and Q67 are located at the active site, but in the consensus structure they are not within hydrogen bonding distance of N14. The hydrogen bond interactions that are observed in X-ray structures of LMW PTPs may in fact be transient in solution. Protein dynamics within the active site hydrogen bonding network appear to be affected by the presence of substrate or bound inhibitors such as inorganic phosphate. 相似文献
17.
The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Psis) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Psi formation in 5S and 5.8S rRNA in which the unassigned Psis occur. We show that while the formation of the Psi in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Psi in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Psi in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis. 相似文献
18.
Three modifications in the D and T arms of tRNA influence translation in Escherichia coli and expression of virulence genes in Shigella flexneri
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The modified nucleosides 2'-O-methylguanosine, present at position 18 (Gm18), 5-methyluridine, present at position 54 (m(5)U54), and pseudouridine, present at position 55 (Psi55), are located in the D and T arms of tRNAs and are close in space in the three-dimensional (3D) structure of this molecule in the bacterium Escherichia coli. The formation of these modified nucleosides is catalyzed by the products of genes trmH (Gm18), trmA (m(5)U54), and truB (Psi55). The combination of trmH, trmA, and truB mutations resulting in lack of these three modifications reduced the growth rate, especially at high temperature. Moreover, the lack of three modified nucleotides in tRNA induced defects in the translation of certain codons, sensitivity to amino acid analog 3,4-dehydro-DL-proline, and an altered oxidation of some carbon compounds. The results are consistent with the suggestion that these modified nucleosides, two of which directly interact in the 3D structure of tRNA by forming a hydrogen bond between Psi55 and Gm18, stabilize the structure of the tRNA. Moreover, lack of Psi55 in tRNA of human pathogen Shigella flexneri leads to a reduced expression of several virulence-associated genes. 相似文献
19.
Small nuclear RNAs (snRNA), cofactors in the splicing of pre-mRNA, are highly modified. In this report the modification of human U4 RNA was studied using cell extracts and in vitro synthesized, and therefore unmodified, U4 RNA. The formation of pseudouridine (Psi) at positions 4, 72 and 79 in U4 RNA was dependent on an RNA-containing cofactor, since the activities in the extracts were micrococcal nuclease (MN) sensitive. Extracts were fractionated on glycerol gradients and there was a broad peak of reconstitution activity centered at 14 S. Reconstitution was not due to additional enzymatic activity, since the peak fraction was MN sensitive. Oligodeoxynucleotide-mediated RNase H digestion of U6 RNA in the extracts inhibited formation of Psi in U4 RNA. From glycerol gradient analysis we determined that exogenously added U4 RNA that is associated with U6 RNA (sedimentation velocity 16 S) was significantly higher in Psi content than U4 RNA not associated with U6 RNA (8 S). Competitive inhibitors of Psi synthases, 5-fluorouridine-containing (5-FU) wild-type and mutant U4 RNAs, were used to investigate formation of Psi in U4 RNA. Deletions and point mutations in these 5-FU-containing U4 RNAs affected their ability to inhibit Psi synthase in vitro. With the aid of these potent inhibitors it was determined that at least two separate activities modify the uridines at these positions. 相似文献
20.
The Saccharomyces cerevisiae Pus2 protein encoded by YGL063w ORF is a mitochondrial tRNA:Psi27/28-synthase
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The RNA:pseudouridine (Psi)-synthase family is one of the most complex families of RNA modification enzymes. Ten genes encoding putative RNA:Psi-synthases have been identified in S. cerevisiae. Most of the encoded enzymes have been characterized experimentally. Only the putative RNA:Psi-synthase Pus2p (encoded by the YGL063w ORF) had no identified substrate. Here, we analyzed Psi residues in cytoplasmic and mitochondrial tRNAs extracted from S. cerevisiae strains, carrying disruptions in the PUS1 and/or PUS2 ORFs. Our results demonstrate that Pus2p is a mitochondrial-specific tRNA:Psi-synthase acting at positions 27 and 28 in tRNAs. The importance of the Asp56 residue in the conserved ARTD motif of the Pus2p catalytic site is demonstrated in vivo. Interestingly, in spite of the absence of a characteristic N-terminal targeting signal, our data strongly suggest an efficient and rapid targeting of Pus2p in yeast mitochondria. In contradiction with the commonly held idea that a unique nuclear gene encodes the enzyme required for both cytoplasmic and mitochondrial tRNA modifications, here we show the existence of an enzyme specifically dedicated to mitochondrial tRNA modification (Pus2p), the corresponding modification in cytoplasmic tRNAs being catalyzed by another protein (Pus1p). 相似文献