8 research outputs found

    The conserved histone deacetylase Rpd3 and the DNA binding regulator Ume6 repress BOI1's meiotic transcript isoform during vegetative growth in Saccharomyces cerevisiae.

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    International audienceBOI1 and BOI2 are paralogs important for the actin cytoskeleton andpolar growth. BOI1 encodes a meiotic transcript isoform with an extended5-untranslated region predicted to impair protein translation. It is,however, unknown how the isoform is repressed during mitosis, and ifBoi1 is present during sporulation. By interpreting microarray data fromMATa cells, MATa/ cells, a starving MAT/ control, and a meiosis-impairedrrp6 mutant, we classified BOI1's extended isoform as earlymeiosis-specific. These results were confirmed by RNA-Sequencing, andextended by a 5-RACE assay and Northern blotting, showing that meioticcells induce the long isoform while the mitotic isoform remainsdetectable during meiosis. We provide evidence via motif predictions, anin vivo binding assay and genetic experiments that the Rpd3/Sin3/Ume6histone deacetylase complex, which represses meiotic genes duringmitosis, also prevents the induction of BOI1's 5-extended isoform inmitosis by direct binding of Ume6 to its URS1 target. Finally, we findthat Boi1 protein levels decline when cells switch from fermentation torespiration and sporulation. The histone deacetylase Rpd3 is conserved,and eukaryotic genes frequently encode transcripts with variable 5-UTRs.Our findings are therefore relevant for regulatory mechanisms involvedin the control of transcript isoforms in multi-cellular organisms

    Structure of photosystem II and substrate binding at room temperature

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    Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment-protein complex, couples the one-electron photochemistry at the reaction center with the four-electron redox chemistry of water oxidation at the Mn(4)CaO(5) cluster in the oxygen-evolving complex (OEC) (Fig. 1a, Extended Data Fig. 1). Under illumination, the OEC cycles through five intermediate S-states (S(0) to S(4))(1), where S(1) is the dark stable state and S(3) is the last semi-stable state before O-O bond formation and O(2) evolution(2,3). A detailed understanding of the O-O bond formation mechanism remains a challenge, and elucidating the structures of the OEC in the different S-states, as well as the binding of the two substrate waters to the catalytic site(4-6), is a prerequisite for this purpose. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage free, room temperature (RT) structures of dark-adapted (S(1)), two-flash illuminated (2F; S(3)-enriched), and ammonia-bound two-flash illuminated (2F-NH(3); S(3)-enriched) PS II. Although the recent 1.95 √Ö structure of PS II(7) at cryogenic temperature using an XFEL provided a damage-free view of the S(1) state, RT measurements are required to study the structural landscape of proteins under functional conditions(8,9), and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analog, has been used as a marker, as it binds to the Mn(4)CaO(5) cluster in the S(2) and S(3) states(10). Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site(10-13). Thus, this approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms


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