The overall goal of this revised proposal is to investigate the protein-nucleic acid interactions which define the transcriptional activity of the Arabidopsis and maize Adh genes in response to development and anaerobic stress. This investigator aims to integrate information on chromatin and DNA structures with information on protein- nucleic acid interactions to molecularly define the transcriptional complexes assembled on these genes. During the previous grant period, in vitro and in vivo binding studies by this investigator have defined a number of G-box and half G-box elements which bind proteins during gene activation. Screening of plant extracts for the GBF factors associated with these elements has importantly demonstrated that these factors are present in root tissues expressing Adh as well as tissues not expressing Adh at all (i.e., leaves). In vivo footprints indicate that these GBF proteins are bound to G-box promoter elements only in transcriptionally active tissues. Cloning of cDNAs for protein factors associated with G-boxes has uncovered an interesting family of GF 14 proteins which are associated with G-box protein complexes but not directly bound to the DNA. Dr. Ferl has identified homologies between GF14 proteins and mammalian Ca2+/calmodulin protein kinaseII-dependent activators of Trp and Tyr hydroxylases (14-3-3 proteins) and also the KCIP inhibitors of protein kinase C. This suggests that G14 may serve a regulatory role in Adh activation.GBF proteins have been cloned from both maize and Arabidopsis. Using these cloned G14 and GBF cDNAs, the investigator proposes defining the interrelationships between these factors and their binding sites which exist at different positions in each of the promoters. Grouping of these factors into classes on the basis of DNase I and DMS footprints and mobility shifts will determine whether individual proteins recognize one or multiple promoter sequences and whether the various elements in the three Adh genes are actually recognized by similar sets of proteins. These experiments also aim to define how GF14 interacts with GBF and whether other proteins are associated with this complex. The second goal of this proposal is to monitor the activation state of Adh promoters in vivo by correlating expression in transgenic plants with in vivo footprints. These studies will determine whether critical binding sites defined by in vitro analysis actually change protein binding and expression in vivo.Many of the binding sites are half G-boxes which potentially interact with heterodimeric GBFs and so the final goal of this proposal is to search for additional pairing partners that interact with GBF.
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