We have isolated the gene encoding a methionine-rich 10-kDa zein protein from a lambda EMBL3 maize genomic 'mini' library of the inbred line BSSS-53 and determined its nucleotide sequence. The sequence matches perfectly with a cDNA clone from the inbred line W22 (which has the same restriction fragment length polymorphism as many inbred lines tested) indicating that we have isolated a functional storage protein gene that is very conserved in maize. This comparison also excludes any splicing of any precursor mRNA and therefore any presence of introns. A number of potential regulatory sequences have been located in the flanking regions. The 10-kDa-zein gene represents the last size class in the zein multigene family to be characterized. Its structure allows us now to re-examine the relationship of all the zein proteins and also to compare the structure of a new class of storage proteins that are rich in methionine, an essential amino acid in livestock fodder.

Although allelic sequences can vary extensively, it is generally assumed that each gene in one individual will have an allelic counterpart in another individual of the same species. We report here that this assumption does not hold true in maize. We have sequenced over 100 kb from the bz genomic region of two different maize lines and have found dramatic differences between them. First, the retrotransposon clusters, which comprise most of the repetitive DNA in maize, differ markedly in make-up and location relative to the genes in the bz region. Second, and more importantly, the genes themselves differ between the two lines, demonstrating that genetic microcolinearity can be violated within the same species. Our finding has bearing on the underlying genetic basis of hybrid vigor in maize, and possibly other organisms, and on the measurement of genetic distances.

The membranes of all eukaryotic motile (9 + 2) and immotile primary (9 + 0) cilia harbor channels and receptors involved in sensory transduction (reviewed by). These membrane proteins are transported from the cytoplasm onto the ciliary membrane by vesicles targeted for exocytosis at a point adjacent to the ciliary basal body. Here, we use time-lapse fluorescence microscopy to demonstrate that select GFP-tagged sensory receptors undergo rapid vectorial transport along the entire length of the cilia of Caenorhabditis elegans sensory neurons. Transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 move in ciliary membranes at rates comparable to the intraflagellar transport (IFT) machinery located between the membrane and the underlying axonemal microtubules. OSM-9 motility is disrupted in certain IFT mutant backgrounds. Surprisingly, motility of transient receptor potential polycystin (TRPP) channel PKD-2 (polycystic kidney disease-2), a mechano-receptor, was not detected. Our study demonstrates that IFT, previously shown to be necessary for transport of axonemal components, is also involved in the motility of TRPV membrane protein movement along cilia of C. elegans sensory cells.

Recent studies of the intracellular signaling pathway initiated by ligands of the transforming growth factor beta (TGFbeta) family have provided new insights into how the receptors for such ligands phosphorylate their substrates - the Smads - and how signaling specificity is achieved.

The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins that control numerous cellular and developmental processes in yeast, Drosophila melanogaster, plants, and mammals. Although these proteins bind DNA on their own, they often combine with different cofactors to bind with increased affinity and specificity to their target sites. To understand how this class of proteins functions, we have made a series of alanine substitutions in the MADS box domain of Mcm1 and examined the effects of these mutations in combination with its cofactors that regulate mating in yeast. Our results indicate which residues of Mcm1 are essential for viability and transcriptional regulation with its cofactors in vivo. Most of the mutations in Mcm1 that are lethal affect DNA-binding affinity. Interestingly, the lethality of many of these mutations can be suppressed if the MCM1 gene is expressed from a high-copy-number plasmid. Although many of the alanine substitutions affect the ability of Mcm1 to activate transcription alone or in combination with the alpha 1 and Ste12 cofactors, most mutations have little or no effect on Mcm1-mediated repression in combination with the alpha 2 cofactor. Even nonconservative amino acid substitutions of residues in Mcm1 that directly contact alpha 2 do not significantly affect repression. These results suggest that within the same region of the Mcm1 MADS box domain, there are different requirements for interaction with alpha 2 than for interaction with either alpha1 or Ste12. Our results suggest how a small domain, the MADS box, interacts with multiple cofactors to achieve specificity in transcriptional regulation and how subtle differences in the sequences of different MADS box proteins can influence the interactions with specific cofactors while not affecting the interactions with common cofactors.

Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP beta subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP beta interactions demonstrates that the CarD/RNAP beta association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP beta interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP beta interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding.

The sigma-subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when sigma associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of sigma and the flap domain of the RNAP beta-subunit (beta-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli sigma(70) that have specific effects on the sigma(70) region 4/beta-flap interaction, either weakening or strengthening it. Using these sigma(70) mutants, we demonstrate that the sigma region 4/beta-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the beta-flap, sigma region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of sigma-core interactions.

Metazoan cells are exposed to a multitude of signals, which they integrate to determine appropriate developmental or physiological responses. Although the Hippo pathway was only discovered recently, and our knowledge of Hippo signal transduction is far from complete, a wealth of interconnections amongst Hippo and other signaling pathways have already been identified. Hippo signaling is particularly important for growth control, and I describe how integration of Hippo and other pathways contributes to regulation of organ growth. Molecular links between Hippo signaling and other signal transduction pathways are summarized. Different types of mechanisms for signal integration are described, and examples of how the complex interconnections between pathways are used to guide developmental and physiological growth responses are discussed. Features of Hippo signaling appear to make it particularly well suited to signal integration, including its responsiveness to cell-cell contact and the mediation of its transcriptional output by transcriptional co-activator proteins that can interact with transcription factors of other pathways.

TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP "clamp" region as a regulatory hot spot for both transcription initiation and transcription elongation.

Using fluorescence resonance energy transfer to monitor distances within single molecules of abortively initiating transcription initiation complexes, we show that initial transcription proceeds through a "scrunching" mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA into itself and past its active center. We show further that putative alternative mechanisms for RNAP active-center translocation in initial transcription, involving "transient excursions" of RNAP relative to DNA or "inchworming" of RNAP relative to DNA, do not occur. The results support a model in which a stressed intermediate, with DNA-unwinding stress and DNA-compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of interactions between RNAP and promoter DNA and between RNAP and initiation factors during promoter escape.

The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) in complex with a sigma factor that is essential for promoter recognition and transcription initiation. During early elongation, the stability of interactions between sigma and the remainder of the transcription complex decreases. Nevertheless, there is no mechanistic requirement for release of sigma upon the transition to elongation. Furthermore, sigma can remain associated with RNAP during transcription elongation and influence regulatory events that occur during transcription elongation. Here we demonstrate that promoter-like DNA sequence elements within the initial transcribed region that are known to induce early elongation pausing through sequence-specific interactions with sigma also function to increase the sigma content of downstream elongation complexes. Our findings establish sigma-dependent pausing as a mechanism by which initial transcribed region sequences can influence the composition and functional properties of the transcription elongation complex over distances of at least 700 base pairs.

We define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to but not overlapping the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight-bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents.

Compartment boundaries play key roles in tissue organization by separating cell populations. Activation of the Notch receptor is required for dorsoventral (DV) compartmentalization of the Drosophila wing, but the nature of its requirement has been controversial. Here, we provide additional evidence that a stripe of Notch activation is sufficient to establish a sharp separation between cell populations, irrespective of their dorsal or ventral identities. We further find that cells at the DV compartment boundary are characterized by a distinct shape, a smooth interface, and an accumulation of F-actin at the adherens junction. Genetic manipulation establishes that a stripe of Notch activation is both necessary and sufficient for this DV boundary cell phenotype, and supports the existence of a non-transcriptional branch of the Notch pathway that influences F-actin. Finally, we identify a distinct requirement for a regulator of actin polymerization, capulet, in DV compartmentalization. These observations imply that Notch effects compartmentalization through a novel mechanism, which we refer to as a fence, that does not depend on the establishment of compartment-specific cell affinities, but does depend on the organization of the actin cytoskeleton.

The Drosophila optic lobe develops from neuroepithelial cells, which function as symmetrically dividing neural progenitors. We describe here a role for the Fat-Hippo pathway in controlling the growth and differentiation of Drosophila optic neuroepithelia. Mutation of tumor suppressor genes within the pathway, or expression of activated Yorkie, promotes overgrowth of neuroepithelial cells and delays or blocks their differentiation; mutation of yorkie inhibits growth and accelerates differentiation. Neuroblasts and other neural cells, by contrast, appear unaffected by Yorkie activation. Neuroepithelial cells undergo a cell cycle arrest before converting to neuroblasts; this cell cycle arrest is regulated by Fat-Hippo signaling. Combinations of cell cycle regulators, including E2f1 and CyclinD, delay neuroepithelial differentiation, and Fat-Hippo signaling delays differentiation in part through E2f1. We also characterize roles for Jak-Stat and Notch signaling. Our studies establish that the progression of neuroepithelial cells to neuroblasts is regulated by Notch signaling, and suggest a model in which Fat-Hippo and Jak-Stat signaling influence differentiation by their acceleration of cell cycle progression and consequent impairment of Delta accumulation, thereby modulating Notch signaling. This characterization of Fat-Hippo signaling in neuroepithelial growth and differentiation also provides insights into the potential roles of Yes-associated protein in vertebrate neural development and medullablastoma.

The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A1A2A3T4G5T6G7A8T9C10T11 -3' ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of approximately 42 degrees, twist angles of approximately 16 degrees ), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of approximately 20 degrees, twist angles of approximately 17 degrees). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, approximately 46 degrees per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, approximately 28 degrees per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.

The catabolite activator protein (CAP) makes no direct contact with the consensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specificity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at position 7 form a T:A/G:C step, which is known to be associated with DNA flexibility. It has been proposed that specificity for T:A at position 6 is a consequence of formation of the DNA kink between positions 6 and 7, and of effects of the T:A(6)/G:C(7) step on the geometry of DNA kinking, or the energetics of DNA kinking. In this work, we determine crystallographic structures of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes containing T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this system does not involve differences in the geometry of DNA kinking but, rather, solely differences in the energetics of DNA kinking. We further infer that the main determinant of DNA conformation in this system is protein-DNA interaction, and not DNA sequence.

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), through an "indirect-readout" mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3' to the primary-kink site, G:C(7), through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C(7). Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A(6) to specificity for C:G(6), and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181-->Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A(6). The Glu181-->Asp substitution does not eliminate hydrogen-bond formation with G:C(7), and thus does not eliminate direct-readout-based specificity for G:C(7).

The amino acid methionine is a common protein building block that is also important in other cellular processes. Plants, unlike animals, synthesize methionine de novo and are thus a dietary source of this nutrient. A new approach for using maize as a source of nutrient methionine is described. Maize seeds, a major component of animal feeds, have variable levels of protein-bound methionine. This variability is a result of post-transcriptional regulation of the Dzs10 gene, which encodes a seed-specific high-methionine storage protein. Here we eliminate methionine variability by identifying and replacing the cis-acting site for Dzs10 regulation using transgenic seeds. Interestingly, two different mechanisms affect mRNA accumulation, one dependent on and the other independent of the untranslated regions (UTRs) of Dzs10 RNA. Accumulation of chimeric Dzs10 mRNA was not reduced in hybrid crosses and was uncoupled from genomic imprinting by Dzr1, a regulator of Dzs10. Uniform high levels of Dzs10 protein were maintained over five backcross generations of the transgene. The increased level of methionine in these transgenic seeds allowed the formulation of a useful animal feed ration without the addition of synthetic methionine.

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O2 evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O2 evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.

We have developed a simple procedure to incorporate an EDTA-metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step 1, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step 2, we derivatize the resulting protein with S-(2-pyridylthio)cysteaminyl-EDTA-metal, a novel aromatic disulfide derivative of EDTA-metal. We have used this procedure to incorporate an EDTA-iron complex at amino acid 2 of the helix-turn-helix motif of each of two helix-turn-helix motif sequence-specific DNA binding proteins, catabolite gene activator protein (CAP) and Cro, and we have analyzed EDTA-iron-mediated DNA affinity cleavage by the resulting protein derivatives. The CAP derivative cleaves DNA at base pair 2 of the DNA half-site in the protein-DNA complex, and the Cro derivative cleaves DNA at base pairs -3 to 5 of the DNA half-site in the protein-DNA complex. We infer that amino acid 2 of the helix-turn-helix motif of CAP is close to base pair 2 of the DNA half-site in the CAP-DNA complex in solution and that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -3 to 5 of the DNA half-site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)