Transforming growth factor beta is a multi-functional growth and differentiation factor responsible for regulating many diverse biological processes in both vertebrate and invertebrate species. Among the most dramatic of TGFbeta's effects are those associated with specification of cell fates during development and inhibition of cell cycle progression. The core TGFbeta signaling pathway has now been described using a synergistic combination of genetic and biochemical approaches. Transmembrane receptors with intrinsic protein serine kinase activity bind ligand in the extracellular milieu and then phosphorylate intracellular proteins known as Smads. Phosphorylated Smads form heterooligomers and translocate into the nucleus where they can modulate transcriptional responses. More recent studies indicate that many other proteins serve as modulators of Smad activity, and utimately define specific cellular responses to TGFbeta. Here we describe both the simplistic core TGFbeta signaling pathway and the growing number of proteins that impinge on this pathway at the level of Smad function to either enhance or inhibit TGFbeta responses.

CAP-dependent promoters can be divided into classes based on the position of the DNA site for CAP. In class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for polymerase; the DNA site for CAP can be located at various distances from the transcription start point, provided that the DNS site for CAP and the DNA site for RNA polymerase are on the same face of the DNA helix. In class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 determinants for binding of RNA polymerase. In previous work, we have shown that a surface loop consisting of amino acid residues 152 to 166 of CAP is essential for transcription activation at the best-characterized class I CAP-dependent promoter, the lac promoter, and we proposed that this surface loop makes direct protein-protein contact with RNA polymerase in the ternary complex of lac promoter, CAP, and RNA polymerase. Here, we show that the surface loop consisting of amino acid residues 152 to 166 is essential for transcription activation at other class I CAP-dependent promoters and at a class II CAP-dependent promoter. We show further that the effects of alanine substitutions of residues 152 to 166 are qualitatively identical at the lac promoter and other class I CAP-dependent promoters, but are different at a class II CAP-dependent promoter. We propose that the surface loop consisting of residues 152 to 166 makes identical molecular interactions in transcription activation at all class I CAP-dependent promoters, irrespective of distance between the DNA site for CAP and the transcription start point, but makes a different set of molecular interactions in transcription activation at class II CAP-dependent promoters.

We have constructed heterodimers consisting of two subunits: one CAP subunit that has a nonfunctional activating region but wild-type DNA binding specificity, and one CAP subunit that has a functional activating region but non-wild-type DNA binding specificity. We have oriented the heterodimers on lac promoter DNA by use of promoter derivatives that have DNA sites for CAP consisting of one wild-type half site and one non-wild-type half site, and we have analyzed the abilities of the oriented heterodimers to activate transcription. Our results indicate that transcription. Our results indicate that transcription activation requires the activating region of only one subunit of CAP: the promoter-proximal subunit. The oriented heterodimers method of this report should be generalizable to other dimeric transcription activator proteins.

We have isolated 21 mutants of catabolite gene activator protein (CAP) defective in transcription activation at the lac promoter but not defective in DNA binding. The amino acid substitutions in the mutants map to a single region of CAP: amino acids 156-162. As assessed in vitro, the substituted CAP variants are nearly completely unable to activate transcription at the lac promoter but bind to DNA with the same affinity and bend DNA to the same extent as wild-type CAP. Our results establish that amino acids 156-162 are critical for transcription activation at the lac promoter but not for DNA binding and DNA bending. In the structure of CAP, amino acids 156-162 are part of a surface loop. We propose that this surface loop makes a direct protein-protein contact with RNA polymerase at the lac promoter.

In Class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for RNA polymerase. In Class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 site. We have used an 'oriented heterodimers' approach to identify the functional subunit of CAP at two Class I promoters having different distances between the DNA sites for CAP and RNA polymerase [CC(-61.5) and CC(-72.5)] and at one Class II promoter [CC(-41.5)]. Our results indicate that transcription activation at Class I promoters, irrespective of the distance between the DNA sites for CAP and RNA polymerase, requires the activating region of the promoter-proximal subunit of CAP. In striking contrast, our results indicate that transcription activation at Class II promoters requires the activating region of the promoter-distal subunit of CAP.

The MATalpha2 (alpha2) repressor interacts with the Mcm1 protein to turn off a-cell type-specific genes in the yeast Saccharomyces cerevisiae. We compared five natural alpha2-Mcm1 sites with an alpha2-Mcm1 symmetric consensus site (AMSC) for their relative strength of repression and found that the AMSC functions slightly better than any of the natural sites. To further investigate the DNA binding specificity of alpha2 in complex with Mcm1, symmetric substitutions at each position in the alpha2 half-sites of AMSC were constructed and assayed for their effect on repression in vivo and DNA binding affinity in vitro. As expected, substitutions at positions in which there are base-specific contacts decrease the level of repression. Interestingly, substitutions at other positions, in which there are no apparent base-specific contacts made by the protein in the alpha2-DNA co-crystal structure, also significantly decrease repression. As an alternative method to examining the DNA binding specificity of alpha2, we performed in vitro alpha2 binding site selection experiments in the presence and absence of Mcm1. In the presence of Mcm1, the consensus sequences obtained were extended and more closely related to the natural alpha2 sites than the consensus sequence obtained in the absence of Mcm1. These results demonstrate that in the presence of Mcm1 the sequence specificity of alpha2 is extended to these positions.

The alpha2 and Mcm1 proteins bind DNA as a heterotetramer to repress transcription of cell-type-specific genes in the yeast Saccharomyces cerevisiae. Based on the DNA sequence requirements for binding by the alpha2-Mcm1 complex, we have searched the yeast genome for all potential alpha2-Mcm1 binding sites. Genes adjacent to the sites were examined for expression in the different cell mating types. These sites were further analyzed by cloning the sequences into a heterologous promoter and assaying for alpha2-Mcm1-dependent repression in vivo and DNA-binding affinity in vitro. Fifty-nine potential binding sites were identified in the search. Thirty-seven sites are located within or downstream of coding region of the gene. None of the sites assayed from this group are functional repressor sites in vivo or bound by the alpha2-Mcm1 complex in vitro. Among the remaining 22 sites, six are in the promoters of known alpha-specific genes and two other sites have an alpha2-Mcm1-dependent role in determining the direction of mating type switching. Among the remaining sequences, we have identified a functional site located in the promoter region of a previously uncharacterized gene, SCYJL170C. This site functions to repress transcription of a heterologous promoter and the alpha2-Mcm1 complex binds to the site in vitro. SCYJL170C is repressed by alpha2-Mcm1 in vivo and therefore using this method we have identified a new a-specific gene, which we call ASG7.

Regulated membrane trafficking of AMPA-type glutamate receptors (AMPARs) is a key mechanism underlying synaptic plasticity, yet the pathways used by AMPARs are not well understood. In this paper, we show that the AMPAR subunit GLR-1 in Caenorhabditis elegans utilizes the retrograde transport pathway to regulate AMPAR synaptic abundance. Mutants for rab-6.2, the retromer genes vps-35 and snx-1, and rme-8 failed to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. In contrast, expression of constitutively active RAB-6.2 drove the retrograde transport of GLR-1 from dendrites back to cell body Golgi. We also find that activated RAB-6.2 bound to and colocalized with the PDZ/phosphotyrosine binding domain protein LIN-10. RAB-6.2 recruited LIN-10. Moreover, the regulation of GLR-1 transport by RAB-6.2 required LIN-10 activity. Our results demonstrate a novel role for RAB-6.2, its effector LIN-10, and the retromer complex in maintaining synaptic strength by recycling AMPARs along the retrograde transport pathway.

The Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein. CAP contains a unique solvent-accessible cysteine residue at amino acid 10 of the helix-turn-helix motif. In published work, we have constructed a prototype semi-synthetic site-specific DNA cleavage agent from CAP by use of cysteine-specific chemical modification to incorporate a nucleolytic chelator-metal complex at amino acid 10 of the helix-turn-helix motif [Ebright, R., Ebright, Y., Pendergrast, P.S. and Gunasekera, A., Proc. Natl. Acad. Sci. USA 87, 2882-2886 (1990)]. Construction of second-generation semi-synthetic site-specific DNA cleavage agents from CAP requires the construction of derivatives of CAP having unique solvent-accessible cysteine residues at sites within CAP other than amino acid 10 of the helix-turn-helix motif. In the present work, we have constructed and characterized two derivatives of CAP having no solvent-accessible cysteine residues: [Ser178]CAP and [Leu178]CAP. In addition, in the present work, we have constructed and characterized one derivative of CAP having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif: [Cys170;Ser178]CAP.

We have used site-directed mutagenesis to replace amino acid 1 of the recognition alpha-helix of the catabolite gene activator protein (CAP), Arg-180, with glycine and with alanine. Substitution of Arg-180 of CAP eliminated specificity between G.C, A.T, C.G, and T.A at base pair 5 of the DNA half-site. The effect was position-specific: substitution of Arg-180 did not eliminate specificity between G.C, A.T, C.G, and T.A at base pair 7 of the DNA half-site. We conclude, in agreement with the model for the structure of the CAP-DNA complex [Weber, I. & Steitz, T. (1984) Proc. Natl. Acad. Sci. USA 81, 3973-3977; and Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Proc. Natl. Acad. Sci. USA 81, 7274-7278], that Arg-180 of CAP makes a specificity-determining contact with base pair 5 of the DNA half-site in the CAP-DNA complex. The identification of the contact by Arg-180 in this report, in conjunction with the identification of the contact by Glu-181 in a previous report [Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Nature (London) 311, 232-235], provides information sufficient to define the orientation of the helix-turn-helix motif of CAP with respect to DNA in the CAP-DNA complex.

During transcription initiation, RNA polymerase (RNAP) binds and unwinds promoter DNA to form an RNAP-promoter open complex. We have determined crystal structures at 2.9 and 3.0 Å resolution of functional transcription initiation complexes comprising Thermus thermophilus RNA polymerase, σ(A), and a promoter DNA fragment corresponding to the transcription bubble and downstream dsDNA of the RNAP-promoter open complex. The structures show that σ recognizes the -10 element and discriminator element through interactions that include the unstacking and insertion into pockets of three DNA bases, and that RNAP recognizes the -4/+2 region through interactions that include the unstacking and insertion into a pocket of the +2 base. The structures further show that interactions between σ and template-strand ssDNA preorganize template-strand ssDNA to engage the RNAP active center.

It has been suggested that the catabolite gene activator protein (CAP) uses an "acidic activating region" transcription activation mechanism and that Glu171 of CAP is the critical amino acid of the "acidic activating region" of CAP (Irwin, N., and Ptashne, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8315-8319). In this paper, we show, contrary to the previously published report, that substitution of Glu171 of CAP fails to result in a specific defect in transcription activation at the lac promoter. Furthermore, in this paper, we show that substitution of each other negatively charged amino acid of CAP that is solvent-accessible in the CAP-DNA complex fails to result in a specific defect in transcription activation at the lac promoter. We conclude that CAP does not use an acidic activating region transcription activation mechanism in transcription activation at the lac promoter.

The consensus DNA site for Escherichia coli catabolite gene activator protein (CAP) is 5'-AAATGTGATCTAGATCACATTT-3'. The proposed consensus DNA site for E. coli FNR is 5'-AAATTTGATATATATCAAATTT-3'. In this report, we show that substitution of 2 base pairs (1 base pair per DNA half-site) within the E. coli lac DNA site for CAP suffices to remove the lac promoter from the CAP regulon and to place the lac promoter in the FNR regulon. FNR stimulates transcription of the derivative of the lac promoter having G:C----T:A substitutions at base pair 5 each DNA half-site (13-fold stimulation). FNR does not stimulate transcription of the wild-type lac promoter, or of derivatives of the lac promoter having G:C----A:T or G:C----C:G substitutions at base pair 5 of each DNA half-site. Stimulation of transcription is strictly dependent on anaerobiosis. FNR-stimulated transcription initiates at the same base pair as does CAP-dependent transcription of the wild-type lac promoter.