tRNA pseudouridine synthase I catalyzes the conversion of uridine to pseudouridine at positions 38, 39, and/or 40 in the anticodon loop of many tRNAs. Pseudouridine synthase I was cloned behind a T7 promoter and expressed in Escherichia coli to about 20% of total soluble proteins. Fluorouracil-substituted tRNA caused a time-dependent inactivation of pseudouridine synthase I and formed a covalent complex with the enzyme that involved the FUMP at position 39. Asp60, conserved in all known and putative pseudouridine synthases, was mutated to amino acids with diverse side chains. All Asp60 mutants bound tRNA but were catalytically inactive and failed to form covalent complexes with fluorouracil-substituted tRNA. We conclude that the conserved Asp60 is essential for pseudouridine synthase activity and propose mechanisms which involve this residue in important catalytic roles.

We developed a transcript profiling methodology to elucidate expression patterns of the cyanobacterium Synechocystis sp. strain PCC 6803 and used the technology to investigate changes in gene expression caused by irradiation with either intermediate-wavelength UV light (UV-B) or high-intensity white light. Several families of transcripts were altered by UV-B treatment, including mRNAs specifying proteins involved in light harvesting, photosynthesis, photoprotection, and the heat shock response. In addition, UV-B light induced the stringent response in Synechocystis, as indicated by the repression of ribosomal protein transcripts and other mRNAs involved in translation. High-intensity white light-and UV-B-mediated expression profiles overlapped in the down-regulation of photosynthesis genes and induction of heat shock response but differed in several other transcriptional processes including those specifying carbon dioxide uptake and fixation, the stringent response, and the induction profile of the high-light-inducible proteins. These two profile comparisons not only corroborated known physiological changes but also suggested coordinated regulation of many pathways, including synchronized induction of D1 protein recycling and a coupling between decreased phycobilisome biosynthesis and increased phycobilisome degradation. Overall, the gene expression profile analysis generated new insights into the integrated network of genes that adapts rapidly to different wavelengths and intensities of light.While sunlight provides the energy for life, intermediatewavelength UV light (UV-B) and long-wavelength UV light (UV-A) injure many organisms. UV-B (280-to 320-nm-wavelength) light generates radicals that damage proteins, nucleic acids, lipids, and the photosynthetic apparatus. The latter damage appears in the form of impaired photosystems I and II, decreased oxygen evolution and CO 2 fixation, inactivation of ATPase activity, reduction in chlorophyll content, and decreased biomass (33). Notably, the D1 protein of the light energy converting complex photosystem II is quite sensitive to UV-B light (10). Although less harmful than UV-B irradiation, UV-A irradiation (320-to 400-nm-wavelength light) also damages proteins, nucleic acids, lipids, and photosystems (11). UV-A irradiation leads to the intramolecular cross-linking of several tRNA species (2), which results in poor aminoacylation and triggering of the stringent response-a phenomenon that reduces the rate of stable RNA, ribosome, and translation factor synthesis, thus arresting growth (32). Higher fluxes of white light also cause a loss of photosynthetic productivity (photoinhibition) (30).In cyanobacteria, more than 99% of UV-B is absorbed by chlorophyll-binding proteins and the light-harvesting complexes (phycobilisomes) (20). Carotenoids protect cells against photooxidative damage by absorbing triplet state energy from chlorophyll and quenching singlet state oxygen (15). In response to changes in light quantity or quality, cyanobacteria modulate the abundance of ch...

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Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3␤-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.Among the natural pigments in plants, anthocyanins are the largest water-soluble group, found in most fruits, flower petals, and leaves. These fascinating compounds can exist in many structural forms, both simple and complex, governed by physiological regulations and chemical modifications which have profound effects on their stability and colors (13). Among the variety of biological roles, anthocyanins are utilized for the recruitment of pollinators and seed dispersers and in UV protection. Initial interest in the practical applications of brightly colored anthocyanins has stemmed from their potential as replacements for banned dyes because they have no apparent adverse effects on human health (2,5,24). Recently, however, much attention has been drawn to anthocyanin-derived plant products due to their general antioxidant properties (10, 17, 22) and a consistent association between the consumption of diets rich in fruits and vegetables and a lower risk of chronic diseases, including cancer and cardiovascular disease (6, 14). As a result, anthocyanins, currently produced industrially as mixtures from various plant extracts, are becoming attractive targets for fermentation production from well-characterized microbial hosts such as Escherichia coli.Six major classes of anthocyanidins, the aglycon forms of anthocyanins, exist: pelargonidin, cyanidin, delphinidin, p...