The structure of the human tRNALys3 anticodon bound to the HIV genome is stabilized by modified nucleosides and adjacent mismatch base pairs

Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNALys3, to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3′-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5′-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNALys3. The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s2U34, and pseudouridine, Ψ39, appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U162•Ψ39 and G163•A38, that maintained a reasonable A-form helix diameter. The tRNA's s2U34 stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Ψ39 stabilized the adjacent mismatched pairs

Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA

Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome’s subunits by binding to Helix 69 (H69) of 23S rRNA. The differential binding of various aminoglycosides to the chemically synthesized terminal domains of the Escherichia coli and human H69 has been characterized using spectroscopy, calorimetry and NMR. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin B and tobramycin than for paromomycin (Kds = 0.3 ± 0.1, 0.2 ± 0.2 and 5.4 ± 1.1 µM, respectively). The binding of streptomycin was too weak to assess. In contrast to the E. coli H69, the human 28S rRNA H69 had a considerable decrease in affinity for the antibiotics, an important validation of the bacterial target. The three conserved pseudouridine modifications (Ψ1911, Ψ1915, Ψ1917) occurring in the loop of the E. coli H69 affected the dissociation constant, but not the stoichiometry for the binding of paromomycin (Kd = 2.6 ± 0.1 µM). G1906 and G1921, observed by NMR spectrometry, figured predominantly in the aminoglycoside binding to H69. The higher affinity of the E. coli H69 for neomycin B and tobramycin, as compared to paromomycin and streptomycin, indicates differences in the efficacy of the aminoglycosides

Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy

The authors of this research article demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle, and show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein. These movements are of crucial importance for understanding the mechanistic principles of this GAD

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

InterCarb: a community effort to improve interlaboratory standardization of the carbonate clumped isotope thermometer using carbonate standards

Increased use and improved methodology of carbonate clumped isotope thermometry has greatly enhanced our ability to interrogate a suite of Earth-system processes. However, interlaboratory discrepancies in quantifying carbonate clumped isotope (Δ47) measurements persist, and their specific sources remain unclear. To address interlaboratory differences, we first provide consensus values from the clumped isotope community for four carbonate standards relative to heated and equilibrated gases with 1,819 individual analyses from 10 laboratories. Then we analyzed the four carbonate standards along with three additional standards, spanning a broad range of δ47 and Δ47 values, for a total of 5,329 analyses on 25 individual mass spectrometers from 22 different laboratories. Treating three of the materials as known standards and the other four as unknowns, we find that the use of carbonate reference materials is a robust method for standardization that yields interlaboratory discrepancies entirely consistent with intralaboratory analytical uncertainties. Carbonate reference materials, along with measurement and data processing practices described herein, provide the carbonate clumped isotope community with a robust approach to achieve interlaboratory agreement as we continue to use and improve this powerful geochemical tool. We propose that carbonate clumped isotope data normalized to the carbonate reference materials described in this publication should be reported as Δ47 (I-CDES) values for Intercarb-Carbon Dioxide Equilibrium Scale

Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania

Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6- biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully