Formation of the conserved pseudouridine at position 55 in archaeal tRNA

Pseudouridine (Ψ) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Ψ55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Ψ55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein

Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding

In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNAiMet. The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-l-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNAiMet, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present

Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus

Pseudouridine at position 55 (Ψ55) in eubacterial tRNA is produced by TruB. To clarify the role of the Ψ55 modification, we constructed a truB gene disruptant (ΔtruB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at 50°C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replaced the truB gene in the genome with mutant genes, which express TruB proteins with very weak or no enzymatic activity. However the growth retardation at 50°C was not rescued by these mutant proteins. Nucleoside analysis revealed that Gm18, m5s2U54 and m1A58 in tRNA from the ΔtruB strain were abnormally increased. An in vitro assay using purified tRNA modification enzymes demonstrated that the Ψ55 modification has a negative effect on Gm18 formation by TrmH. These experimental results show that the Ψ55 modification is required for low-temperature adaptation to control other modified. 35S-Met incorporation analysis showed that the protein synthesis activity of the ΔtruB strain was inferior to that of the wild-type strain and that the cold-shock proteins were absence in the ΔtruB cells at 50°C

Biochemical and Structural Studies of Pseudouridine 55 Synthase

118 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.Sequence alignment of the families of PsiS identifies the conserved aspartic acid discussed previously as the strictly conserved amino acid. Structural alignment identifies the conserved tyrosine, which is part of hydrophobic core in the active site. The role of the conserved tyrosine is less obvious in addition to apparent structural role. Enzymatic activity assay reveals that mutating T. maritima Tyr67 to any other amino acids abolishes the enzymatic activity. Structure of T. maritima Y67F in complex with the 5FU-RNA reveals, however, that the same 5FhPsi product formed by wild-type Psi55S is also found in the active site. Furthermore, HPLC analysis indicates that 5FhPsi is also formed when 5FU RNA was incubated with either E. coli Y76F or Y76L but not with Y76A. The combined information from structural, biochemical and mutational studies allows us to propose the likely role of the conserved tyrosine as a general base for proton abstraction in PsiS-catalyzed reaction as well as structural role of the hydrophobic phenol ring in the active site.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Conformational change of pseudouridine 55 synthase upon its association with RNA substrate

Pseudouridine 55 synthase (Ψ55S) catalyzes isomerization of uridine (U) to pseudouridine (Ψ) at position 55 in transfer RNA. The crystal structures of Thermotoga maritima Ψ55S, and its complex with RNA, have been determined at 2.9 and 3.0 Å resolutions, respectively. Structural comparisons with other families of pseudouridine synthases (ΨS) indicate that Ψ55S may acquire its ability to recognize a stem–loop RNA substrate by two insertions of polypeptides into the ΨS core. The structure of apo-Ψ55S reveals that these two insertions interact with each other. However, association with RNA substrate induces substantial conformational change in one of the insertions, resulting in disruption of interaction between insertions and association of both insertions with the RNA substrate. Specific interactions between two insertions, as well as between the insertions and the RNA substrate, account for the molecular basis of the conformational change

Chemical compositions and biological activities of essential oils obtained from some Apiaceous and Lamiaceous plants collected in Thailand

Objective: To determine the chemical composition, as well as the antioxidant, antityrosinase and antibacterial activities of essential oils obtained from some Apiaceous and Lamiaceous plants collected in Thailand. Methods: The essential oils of the specified spices and aromatic herbs were obtained by hydro-distillation, and their chemical constituents were analyzed by gas chromatography/mass spectrometry. Antioxidant assays were based on the scavenging effects of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) free radicals as well as the lipid oxidation inhibition of ß-carotene bleaching by linoleic acid. Tyrosinase enzyme inhibition was evaluated by the dopachrome method. Broth microdilution technique was performed for the purposes of studying microbial growth inhibition against the isolated bacterial strains. Results: The essential oils of Elsholtzia stachyodes, Coleus amboinicus (I) and Trachyspermum ammi presented a high degree of potency in DPPH, ABTS and ß-carotene bleaching assays. The Trachyspermum ammi oil, which mainly contained thymol (49.04%) and p-cymene (22.06%), proved to be the most effective in terms of antibacterial activity. The major compositions of Coleus amboinicus (I) were carvacrol (51.57%), y-terpinene (18.04%) and p-cymene (7.81%); while thymol (43.76%) and y-terpinene (24.61%) were identified as the major components of Elsholtzia stachyodes oil, with p-cymene (6.73%) being identified as a minor constituent. Moreover, Cuminum cyminum oil containing cuminaldehyde (49.07%) and Elsholtzia communis oil composed with geranial (44.74%) and neral (35.27%) as the major components displayed a specific ability for the inhibition of the mushroom tyrosinase enzyme. Conclusions: The results indicated that these bioactive essential oils obtained from indigenous herbs are of significant interest as alternative raw materials in food, cosmetic and medicinal products

Complete genome sequence of Pseudomonas aeruginosa PA99 clinical isolate from Thailand carrying two novel class 1 integrons, In2083 and In2084

ABSTRACT: Objectives: The aim of this study was to identify and characterize multidrug resistance genes and the genetic contexts of integrons found in extensively drug resistant (XDR) Pseudomonas aeruginosa PA99 clinical isolate from Thailand. Methods: The sequencing of P. aeruginosa PA99 genomic DNA was done by using Pacific Biosciences RS II sequencing platform. The generated reads were de novo assembled by Canu version 1.4 and the annotation was performed using Prokka v1.12b. The complete genome sequence was subjected for identification of sequence type, serotype, integrons, and antimicrobial resistance genes by MLST 2.0, PAst 1.0, INTEGRALL, Resfinder 4.1, and CARD 3.2.5, respectively. Results: Pseudomonas aeruginosa PA99 genome consisted of a 6,946,480-bp chromosomal DNA with 65.9% GC and belonged to ST964 and serotype O4. Twenty-one antimicrobial resistance genes conferring XDR phenotype were identified. Of special note were carbapenem resistance genes (blaIMP-1, blaPAO, blaOXA-21, and blaOXA-396) and colistin resistance gene basR with L71R mutation. Integron analysis revealed that P. aeruginosa PA99 harbored five class 1 integrons: two copies of In994 (blaIMP-1), an In1575 (aadB), and two novel integrons, In2083 (blaOXA-21 - aac(6’)-Ib3 - aac(6’)-Ib-cr - ere(A)1∆2 - dfrA1r) and In2084 (blaIMP-1 - aac(6’)-Ib3 - aac(6’)-Ib-cr). Conclusions: To the best of our knowledge, this is the first report of two novel class I integrons designated by INTEGRALL as In2083 and In2084 found in XDR-P. aeruginosa PA99 clinical isolate from Thailand. The characterization of genetic contexts of In2083 and In2084 provide the evidence of the assorting of resistance genes to evolve as novel integrons