PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine

Pyrrolysine is represented by an amber codon in genes encoding proteins such as the methylamine methyltransferases present in some Archaea and Bacteria. Pyrrolysyl-tRNA synthetase (PylRS) attaches pyrrolysine to the amber-suppressing tRNA^Pyl. Archaeal PylRS, encoded by pylS, has a catalytic C-terminal domain but an N-terminal region of unknown function and structure. In Bacteria, homologs of the N- and C-terminal regions of archaeal PylRS are respectively encoded by pylSn and pylSc. We show here that wild type PylS from Methanosarcina barkeri and PylSn from Desulfitobacterium hafniense bind tRNA^Pyl in EMSA with apparent K_d values of 0.12 and 0.13 μm, respectively. Truncation of the N-terminal region of PylS eliminated detectable tRNA^Pyl binding as measured by EMSA, but not catalytic activity. A chimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNA^Pyl binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNA^Pyl binding. Further indicating the specificity of PylSn interaction with tRNA^Pyl, substitutions of conserved residues in tRNA^Pyl in the variable loop, D stem, and T stem and loop had significant impact in binding, whereas those having base changes in the acceptor stem or anticodon stem and loop still retained the ability to complex with PylSn. PylSn and the N terminus of PylS comprise the protein superfamily TIGR03129. The members of this family are not similar to any known RNA-binding protein, but our results suggest their common function involves specific binding of tRNA^Pyl.     

Proline Biosynthesis Is Required for Endoplasmic Reticulum Stress Tolerance in Saccharomyces cerevisiae

The amino acid proline is uniquely involved in cellular processes that underlie stress response in a variety of organisms. Proline is known to minimize protein aggregation, but a detailed study of how proline impacts cell survival during accumulation of misfolded proteins in the endoplasmic reticulum (ER) has not been performed. To address this we examined in Saccharomyces cerevisiae the effect of knocking out the PRO1, PRO2, and PRO3 genes responsible for proline biosynthesis. The null mutants pro1, pro2, and pro3 were shown to have increased sensitivity to ER stress relative to wild-type cells, which could be restored by proline or the corresponding genetic complementation. Of these mutants, pro3 was the most sensitive to tunicamycin and was rescued by anaerobic growth conditions or reduced thiol reagents. The pro3 mutant cells have higher intracellular reactive oxygen species, total glutathione, and a NADP⁺/NADPH ratio than wild-type cells under limiting proline conditions. Depletion of proline biosynthesis also inhibits the unfolded protein response (UPR) indicating proline protection involves the UPR. To more broadly test the role of proline in ER stress, increased proline biosynthesis was shown to partially rescue the ER stress sensitivity of a hog1 null mutant in which the high osmolality pathway is disrupted.     

聚球藻7942高效泌氨突变种的获得及其泌氨、谷氨酰胺合成酶活性、光合和生长

将含有Ａｎａｂａｅｎａｓｐ．ＰＣＣ７１２０反义ｇｌｎＡ基因片段的穿梭表达质粒ｐＤＣ－ＡＴＧＳ转化单细胞蓝藻聚球藻Ｓｙｎｅ－ｃｈｏｃｏｃｃｕｓ　ｓｐ．ＰＣＣ７９４２,通过同源重组,外源ＤＮＡ定位整合到染色体上。经过抗菌素筛选,获得一种高效泌氨的Ｓｙｎｅｃhｏｃｏｃｃｕｓ　ｓｐ．７９４２突变种。将此突变种固定化在聚氨脂泡沫中后,定量测定其谷氨酰胺合成酶（ＧＳ）活性。结果表明,固定化后的突变藻培养９ｄ后泌氨活性比自由生活的野生藻高１５６倍,ＧＳ活性降低７３．６％;其生长速度与同条件下野生藻相近,７７Ｋ荧光光谱表明突变种固定化后光系统Ⅱ活性提高４４％。     

Establishment of a Highly Efficient Ammonia Secreting Mutant of Synechococcus sp. PCC 7942 and the Glutamine Synthetase Activity,Photosynthesis and Growth in Its Immobilized Cells

A recombinate plasmid pDC-ATGS was constructed, which contained the antisense fragment of glnA gene from Anabaena sp. PCC 7120 and transformed the unicellular cyanobactefium Synechococcus sp. PCC 7942. The foreign DNA was inserted into the site of glnA locus of the chromosome through the homologous recombination. By using neomyisin, a highly efficient ammonia secretion mutant was selected. After immobilized, the cells of the mutant within polyurethane (PU) foams, glutamine synthetase (GS) and NIt4+ secretory activity of GS, and its growth and photosynthesis were measured. It was shown that NH4+ secretion of the immobilized mutant was enhanced 156 folds which was much higher than that of free-living cells of the wild type. The activity of GS was decreased by 73.6%. Growth of the mutant was the same as that of the wild type. The activity of photosystem Ⅱ in the immobilized mutant cells increased by 44% with 77 K fluorescence spectrum measurement.     

Dhrs3 Protein Attenuates Retinoic Acid Signaling and Is Required for Early Embryonic Patterning

All-trans-retinoic acid (atRA) is an important morphogen involved in many developmental processes, including neural differentiation, body axis formation, and organogenesis. During early embryonic development, atRA is synthesized from all-trans-retinal (atRAL) in an irreversible reaction mainly catalyzed by retinal dehydrogenase 2 (aldh1a2), whereas atRAL is converted from all-trans-retinol via reversible oxidation by retinol dehydrogenases, members of the short-chain dehydrogenase/reductase family. atRA is degraded by cytochrome P450, family 26 (cyp26). We have previously identified a short-chain dehydrogenase/reductase 3 (dhrs3), which showed differential expression patterns in Xenopus embryos. We show here that the expression of dhrs3 was induced by atRA treatment and overexpression of Xenopus nodal related 1 (xnr1) in animal cap assay. Overexpression of dhrs3 enhanced the phenotype of excessive cyp26a1. In embryos overexpressing aldh1a2 or retinol dehydrogenase 10 (rdh10) in the presence of their respective substrates, Dhrs3 counteracted the action of Aldh1a2 or Rdh10, indicating that retinoic acid signaling is attenuated. Knockdown of Dhrs3 by antisense morpholino oligonucleotides resulted in a phenotype of shortened anteroposterior axis, reduced head structure, and perturbed somitogenesis, which were also found in embryos treated with an excess of atRA. Examination of the expression of brachyury, not, goosecoid, and papc indicated that convergent extension movement was defective in Dhrs3 morphants. Taken together, these studies suggest that dhrs3 participates in atRA metabolism by reducing atRAL levels and is required for proper anteroposterior axis formation, neuroectoderm patterning, and somitogenesis.     

Genetic and biochemical characterization of a pathway for the degradation of 2-aminoethylphosphonate in Sinorhizobium meliloti 1021

A variety of microorganisms have the ability to use phosphonic acids as sole sources of phosphorus. Here, a novel pathway for degradation of 2-aminoethylphosphonate in the bacterium Sinorhizobium meliloti 1021 is proposed based on the analysis of the genome sequence. Gene deletion experiments confirmed the involvement of the locus containing phnW, phnA, and phnY genes in the conversion of 2-aminoethylphosphonate to inorganic phosphate. Biochemical studies of the recombinant PhnY and PhnA proteins verified their roles as phosphonoacetaldehyde dehydrogenase and phosphonoacetate hydrolase, respectively. This pathway is likely not limited to S. meliloti as suggested by the presence of homologous gene clusters in other bacterial genomes.     

The Src Homology and Collagen A (ShcA) Adaptor Protein Is Required for the Spatial Organization of the Costamere/Z-disk Network during Heart Development

Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca²⁺/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.