Analysis of the Maize Polyubiquitin-1 Promoter Heat Shock Elements and Generation of Promoter Variants with Modified Expression Characteristics

The maize polyubiquitin-1 (Ubi-1) promoter is one of a few select promoters used to express foreign genes in monocots, such that recombinant proteins can be produced at commercially viable levels. Modifying the activity, specificity and responsiveness of such promoters provides a means to achieve desired levels and patterns of expression of genes encoding target products. Ubi-1 is constitutively expressed but is further induced by heat shock. The promoter contains two overlapping sequences with similarity to defined heat shock elements and we show that these sequences are also present upstream of the Ubi-1 homologue isolated from teosinte. Both the maize and teosinte promoters can mediate a heat shock response in transgenic maize. We have dissected the overlapping maize Ubi-1 promoter heat shock elements and demonstrate that the 3' element is required to mediate a heat shock response. The Ubi-1 promoter is particularly active in tissues consisting of rapidly dividing cells, and within the seed it is strongly biased towards driving expression in the embryo. However, replacement of the heat shock elements with a trimer of a basic domain/leucine zipper factor binding site of a pea lectin promoter shifts the balance in seed expression towards the endosperm. The Ubi-1 variants described here differ in their overall activity in the seed, but they all show potential for driving high levels of heterologous gene expression in maize.     

trans-repression of the mouse c-fos promoter: a novel mechanism of Fos-mediated trans-regulation

Fos protein can trans-activate AP-1-dependent gene expression and trans-repress the c-fos promoter. Although we find that trans-repression is enhanced by coexpression of c-Jun, it does not require any of the AP-1 or ATF sites in the mouse c-fos promoter. A major target for repression is the serum response element (SRE). Fos mutants with an impaired leucine zipper are defective in trans-repression and transformation, suggesting that these functions involve the formation of Fos protein complexes. In contrast, mutations that abolish DNA binding of Fos enhance trans-repression but destroy the transforming potential of Fos. In addition, v-Fos protein efficiently transforms but is unable to trans-repress. These findings point to different mechanisms involved in trans-activation and trans-repression and suggest that trans-repression of the type described here is neither sufficient nor required for Fos-induced transformation.     

Dimerization of cGMP-dependent protein kinase 1alpha and the myosin-binding subunit of myosin phosphatase: role of leucine zipper domains

Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase, which dephosphorylates myosin light chains. We recently found that cGMP-dependent protein kinase 1alpha binds directly to the myosin-binding subunit (MBS) of myosin phosphatase via the leucine/isoleucine zipper of cGK. We have now studied the role of the leucine zipper domain of MBS in dimerization with cGK and the leucine/isoleucine zipper and leucine zipper domains of both proteins in homodimerization. Mutagenesis of the MBS leucine zipper domain disrupts cGKIalpha-MBS dimerization. Mutagenesis of the MBS leucine zipper eliminates MBS homodimerization, while similar disruption of the cGKIalpha leucine/isoleucine zipper does not prevent formation of cGK dimers. The MBS leucine zipper domain is phosphorylated by cGK, but this does not have any apparent effect on heterodimer formation between the two proteins. MBS LZ mutants that are unable to bind cGK were poor substrates for cGK. These data support the theory that the MBS leucine zipper domain is necessary and sufficient to mediate both MBS homodimerization and binding of the protein to cGK. In contrast, the leucine/isoleucine zipper of cGK is required for binding to MBS, but not for cGK homodimerization. These data support that the MBS and cGK leucine zipper domains mediate the interaction between these two proteins. The contribution of these domains to both homodimerization and their specific interaction with each other suggest that additional regulatory mechanisms involving these domains may exist.     

Regulatory interfaces between the stress protein response and other gene expression programs in the cell

The stress protein response involves the immediate reprogramming of gene expression in cells exposed to proteomic insult leading to massive synthesis of heat shock proteins (HSP). We have examined the outcome when cells are induced to activate two other gene expression programs--the acute inflammatory response and entry of quiescent cells into the cell cycle--and then exposed to protein stress. We find that these responses are mutually antagonistic with, on the one hand, heat shock factor 1 (HSF1) inhibition through the phosphorylation of inhibitory serine residues after inflammatory or mitogenic stimulus and, on the other hand, after stress, HSF1 directly repressing the promoters of genes that mediate acute inflammation and mitogenesis. The expression of the stress protein response during periods of acute protein damage was shown to lead to efficient activation of HSF1 and HSP expression accompanied by repression of other gene expression programs.     

Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.     

Determination of binding constant of transcription factor AP-1 and DNA. Application of inhibitors.

The equilibrium binding and association kinetics of the fos-jun dimer (basic and leucine zipper domain) to the AP-1 DNA were studied using a quantitative assay. The basic-region and leucine zipper (bZip) domain of c-fos was expressed as a fusion protein with glutathione S-transferase, and it was bound to glutathione-agarose. The GST-fused fos bZip region was allowed to form a heterodimer with the bZip domain of c-jun, to which radiolabeled AP-1 nucleotides were added. After thorough washing, the gel-bound radioactivity was counted. The binding and dissociation rate constants (k(1) and k-(1)) of the fos-jun dimer and DNA could be obtained from a time-course experiment. The association binding constant (K(1)) was determined using both a thermodynamic equation and kinetic parameters. Nordihydroguaiaretic acid (NDGA), momordin I, natural product inhibitors of the fos-jun/DNA complex formation, was applied to this jun-GST-fused fos system and it was found to decrease the apparent equilibrium binding of dimer and DNA. The thermodynamic constant of dimer and inhibitor binding was also determined.