Spectral studies of lactose repressor protein modified with nitrophenol reporter groups.
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Bacterial Proteins↗
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Results for “repressor proteins”
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N-Bromosuccinimide modification of Lac repressor protein.
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Amino Acids↗
Lactose repressor protein modified with fluorescein mercuric acetate.
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Bacterial Proteins↗
Novel replication inhibitory function of the developmental regulator/transcription repressor protein WT1 encoded by the Wilms' tumor gene.
The tumor suppressor/developmental regulator protein WT1 encoded by the Wilms' tumor gene is a zinc finger-containing transcription factor which binds to the G+C-rich motif 5'-GCGGGGGCG-3' and represses transcription. Alternatively spliced variants of WT1 (termed+KTS) having an insertion in the zinc finger region are defective for binding to and hence for repression of transcription from promoters containing this motif. Due to the known interactions of two other tumor suppressor proteins with the simian virus 40 (SV40) oncoprotein large tumor antigen (TAg) [which in one case (p53) results in inhibition of the replication initiation activity of TAg], and because of the presence of G+C-rich sequences in the SV40 origin region, we tested the effect of WT1 on TAg- and SV40 origin-dependent DNA replication. WT1 and its alternatively spliced variants were found to be potent inhibitors of replication. Inhibition of replication by WT1 required portions of the N-terminal transcription repression domain and the C-terminal DNA binding domain, while other WT1 sequences needed for transcriptional regulation were dispensable. WT1 neither inhibited the synthesis of TAg nor formed a stable complex with it. Studies of the requirement of cis-active origin sequences in vivo and protein-DNA interactions in vitro indicated that WT1 and its alternatively spliced variants might inhibit replication by their novel binding to the GC box promoter motifs of the SV40 21 bp repeat replication-auxiliary sequence.
Alternative Splicing↗
Functional characterization of promoter elements involved in regulation of human B beta-fibrinogen expression. Evidence for binding of novel activator and repressor proteins.
A high level of plasma fibrinogen has been shown to be an important risk factor for myocardial infarction and stroke. Thus, we were prompted to investigate regulation of human fibrinogen biosynthesis, a process wherein expression of the B beta-chain of fibrinogen appears to be rate-limiting for fibrinogen secretion. Using electrophoretic mobility shift assays with synthetic probes representing portions of the human B beta-fibrinogen promoter, we have defined several elements that bind distinct classes of transcription factors present in human hepatoma cell nuclear extracts. The contribution of each element to promoter activity was demonstrated in transfection experiments using promoter-chloramphenicol acetyltransferase constructs and human hepatoma cells. Our observations indicate that two distinct sequence elements are required for maximal induction of transcription by interleukin-6. One of these sequences is an IL-6-RE core element similar to that reported for the rat alpha 2-macroglobulin promoter and the other is a binding site for the C/EBP family of transcription factors. We also report two additional elements, one negative- and one positive-acting, that bind novel sequence-specific factors.
Base Sequence↗
Genetic assignment of resonances in the NMR spectrum of a protein: lac repressor.
By using a systematic genetic approach, the resonances in the 19F NMR spectrum of 3-fluorotyrosine-substituted lac repressor protein have been assigned. The NMR data indicate that each monomer of the repressor consists of two distinct and independent domains. One domain, the NH2-terminal sixth of the primary sequence, which has been shown to be very important for DNA binding, is very mobile. The remaining COOH-terminal sequence is more rigid. Ligands of the repressor, which affect its DNA binding capability, lead to conformational changes in the COOH-terminal domain. The approach to the assignment of spectral features taken here can be extended to other systems.
Amino Acids↗
Regulation of the SLT-1A toxin operon by a ferric uptake regulatory protein in toxinogenic strains of Shigella dysenteriae type 1.
The regulation of Shiga toxin expression in a clinical isolate of S. dysenteriae 1 by the Fe-Fur (Iron-ferric uptake regulatory protein) repressor complex was investigated. The presence of an endogenous Fur repressor protein capable of binding to either a Fur binding consensus sequence or the regulatory region of SLT-1A was determined in toxinogenic strains of S. dysenteriae. Plasmid constructs bearing Fur binding sites fused to readily assayable reporter genes were used. Plasmid pSC27.1 contains a 21 bp synthetic oligonucleotide Fur protein binding consensus sequence located upstream to the gene for beta-galactosidase. Plasmid pSC105 contains the regulatory sequences of Shiga-like toxin-1A located upstream to the gene for alkaline phosphatase. In an analogous fashion to Shiga toxin regulation in S. dysenteriae 1, transformants bearing either pSC27.1 or pSC105 plasmid DNA were repressed in gene product expression when grown in minimal medium supplemented with iron. Conversely, transformants were de-repressed when grown under iron limiting conditions. These data suggest the presence of Fe-Fur mediated regulation of toxinogenesis in clinical isolates of S. dysenteriae.
Bacterial Proteins↗
Regulation of methionine synthesis in Escherichia coli: effect of metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL and metJ genes.
The regulation of the expression of three Escherichia coli met genes, metB, which codes for cystathionine gamma-synthetase (EC 4.2.99.9), metL, which codes for aspartokinase II-homoserine dehydrogenase II (EC 2.7.2.4-EC 1.1.1.3) and metJ, which codes for the methionine regulon aporepressor, has been studied using highly purified DNA-directed in vitro protein synthesis systems. In a system where the entire gene product is synthesized, the expression of the metB and metL genes is specifically inhibited by MetJ protein (repressor protein) and S-adenosylmethionine (AdoMet). In a simplified system that measures the formation of the first dipeptide of the gene product (fMet-Ala for the metJ gene), MetJ protein and AdoMet partially repress (approximately 40-60%) metJ gene expression. Thus, the metJ gene can be partially autoregulated by its gene product.
Bacterial Proteins↗
Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 repressor.
DELLA proteins are nuclear repressors of plant gibberellin (GA) responses. Here, we investigate the properties of SLN1, a DELLA protein from barley that is destabilized by GA treatment. Using specific inhibitors of proteasome function, we show that proteasome-mediated protein degradation is necessary for GA-mediated destabilization of SLN1. We also show that GA responses, such as the aleurone alpha-amylase response and seedling leaf extension growth, require proteasome-dependent GA-mediated SLN1 destabilization. In further experiments with protein kinase and protein phosphatase inhibitors, we identify two additional signaling steps that are necessary for GA response and for GA-mediated destabilization of SLN1. Thus, GA signaling involves protein phosphorylation and dephosphorylation steps and promotes the derepression of GA responses via proteasome-dependent destabilization of DELLA repressors.
Alleles↗
Epidermal growth factor enhances insulin-like growth factor binding protein-1 synthesis in human hepatoma cells.
Epidermal growth factor (EGF) was found to induce a rapid 2-fold increase in the amount of insulin-like growth factor binding protein-1 (IGFBP-1) mRNA in human hepatoma Hep2G cells, and this was accompanied by a 2-fold increase in IGFBP-1 secretion. A protein synthesis inhibitor cycloheximide (CHX) caused a 2-3-fold increase in the amount of IGFBP-1 mRNA, which could be accounted for the observed stabilization in decay of IGFBP-1 mRNA after CHX treatment. In nuclear run-on transcription experiments neither EGF nor CHX affected the transcription rate of the IGFBP-1 gene. It is concluded that EGF increases IGFBP-1 secretion rapidly by enhancing IGFBP-1 mRNA accumulation, and the addition of a protein synthesis inhibitor results in a specific increment of IGFBP-1 mRNA, suggesting that a labile protein repressor protein is involved in the turnover IGFBP-1 mRNA.
Blotting, Northern↗
Regulation of methionine synthesis in Escherichia coli: Effect of metJ gene product and S-adenosylmethionine on the expression of the metF gene.
The regulation of the expression of the Escherichia coli metF gene, which codes for 5,10-methylenetet-rahydrofolate reductase (EC 1.1.99.15), has been investigated by using a simplified DNA-directed in vitro system that measures the formation of the first dipeptide (fMet-Ser) of the gene product. The synthesis of fMet-Ser directed by a plasmid containing the metF gene is specifically inhibited by metJ protein (repressor protein). S-Adenosylmethionine enhances the inhibition by the metJ protein of metF gene expression. The inhibition by the metJ protein is at the level of transcription and the results suggest that S-adenosylmethionine is functioning as an allosteric effector.
Journal Article↗
dCtBP-dependent and -independent repression activities of the Drosophila Knirps protein.
Transcriptional repressor proteins play essential roles in controlling the correct temporal and spatial patterns of gene expression in Drosophila melanogaster embryogenesis. Repressors such as Knirps, Krüppel, and Snail mediate short-range repression and interact with the dCtBP corepressor. The mechanism by which short-range repressors block transcription is not well understood; therefore, we have undertaken a detailed structure-function analysis of the Knirps protein. To provide a physiological setting for measurement of repression, the activities of endogenous or chimeric Knirps repressor proteins were assayed on integrated reporter genes in transgenic embryos. Two distinct repression functions were identified in Knirps. One repression activity depends on dCtBP binding, and this function maps to a C-terminal region of Knirps that contains a dCtBP binding motif. In addition, an N-terminal region was identified that represses in a CtBP mutant background and does not bind to the dCtBP protein in vitro. Although the dCtBP protein is important for Knirps activity on some genes, one endogenous target of the Knirps protein, the even-skipped stripe 3 enhancer, is not derepressed in a CtBP mutant. These results indicate that Knirps can utilize two different pathways to mediate transcriptional repression and suggest that the phenomenon of short-range repression may be a combination of independent activities.
Alcohol Oxidoreductases↗
Perturbation of lac operator DNA modification by tryptic core protein from lac repressor.
Operator DNA fragments were modified in the presence of lac repressor protein or its trypsin-resistant core. Operator DNA was alkylated or cleaved enzymatically with these related proteins present to compare the influences of their binding on the reactivities or enzymatic susceptibilities of individual bases in the sequence. These two protein species have pronounced and distinguishable effects on the reactivity of the bases of the operator fragment toward methylation by dimethyl sulfate. Perturbation of base alkylation by the trypsin-resistant core repressor is most pronounced in the inner, asymmetric region of the operator DNA, while repressor effects extend further on either end of the operator sequence. Digestion of the two protein-operator complexes by DNase I yields fragment patterns that differ primarily in extent of protection. These data extend the experimental base supporting the involvement of the core region of the lac repressor in addition to its NH2 termini in the operator-specific binding activity of this protein.
DNA, Bacterial↗
Interaction of yeast repressor-activator protein Ume6p with glycogen synthase kinase 3 homolog Rim11p.
Meiosis and expression of early meiotic genes in the budding yeast Saccharomyces cerevisiae depend upon Rim11p, Ume6p, and Ime1p. Rim11p (also called Mds1p and ScGSK3) is a protein kinase related to glycogen synthase kinase 3 (GSK3); Ume6p is an architectural transcription factor; and Imelp is a Ume6p-binding protein that provides a transcriptional activation domain. Rim11p is required for Ime1p-Ume6p interaction, and prior studies have shown that Rim11p binds to and phosphorylates Ime1p. We show here that Rim11p binds to and phosphorylates Ume6p, as well. Amino acid substitutions in Ume6p that alter a consensus GSK3 site reduce or abolish Rim11p-Ume6p interaction and Rim11p-dependent phosphorylation, and they cause defects in interaction between Ume6p and Ime1p and in meiotic gene expression. Therefore, interaction between Rim11p and Ume6p, resulting in phosphorylation of Ume6p, is required for Ime1p-Ume6p complex formation. Rim11p, like metazoan GSK3beta, phosphorylates both interacting subunits of a target protein complex.
Base Sequence↗
Structure of the DNA-binding region of lac repressor inferred from its homology with cro repressor.
It is shown that the amino acid sequence and the DNA gene sequence of the 25 amino-terminal residues of the lac repressor protein of Escherichia coli are homologous with the sequences of five DNA-binding proteins: the cro repressor proteins from phage lambda and phage 434, the cI and cII proteins from phage lambda, and the repressor protein from Salmonella phage P22. The region of homology between lac repressor and the other proteins coincides with the principal DNA-binding region of cro repressor. In particular, residues Tyr-17 through Gln-26 of lac repressor correspond to the alpha-helix Gln-27 through Ala-36 of cro repressor, which we have postulated to bind within the major groove of the DNA and to be primarily responsible for the recognition of the DNA operator region by the protein [Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews, B. W. (1981) Nature (London) 290, 754--758]. By analogy with cro repressor, we propose that residues 17--26 of lac repressor are alpha-helical and that this helix and a twofold-related alpha-helix in an adjacent subunit bind within successive major grooves of the lac operator, which is in a right-handed Watson--Crick B-DNA conformation. Also, by analogy with cro repressor, we suggest that residues Thr-5 through Ala-13 of lac repressor form a second alpha-helix and contribute, in part, to DNA binding. The proposed structure for the DNA-binding region of lac repressor is consistent with chemical protection data and with genetic experiments identifying the probable locations of a number of the residues of the repressor protein that either do or do not participate in DNA binding.
Binding Sites↗
Unusual mRNA pseudoknot structure is recognized by a protein translational repressor.
Translation of ribosomal proteins in the alpha operon of E. coli is repressed by one of the encoded proteins, S4; it specifically recognizes an RNA fragment containing the translational initiation site for the first gene in the operon. RNA structure mapping experiments have suggested a pseudoknot structure for the S4 binding site: the loop of a hairpin is base paired to sequences downstream of the hairpin. Here, we systematically test this proposed structure by measuring S4 binding to an extensive set of site-directed mutations that create compensatory base pair changes in potential helices. The pseudoknot folding is confirmed, and two additional, unexpected interactions within the pseudoknot are also detected. The overall structure is an unusual "double pseudoknot" linking a hairpin upstream of the ribosome binding site with sequences 2-10 codons downstream of the initiation codon. Stabilization of this structure by S4 could account for translational repression.
Base Composition↗
Thermo-inducible expression of a recombinant fusion protein by Escherichia coli lac repressor mutants.
The lac I gene of Escherichia coli encodes the lactose repressor. We isolated temperature-sensitive mutants of the lac I gene by in vitro mutagenesis with hydroxylamine. The new mutation sites were determined, and replacement of a single amino acid had respectively occurred at amino acid positions 241 (Ala-->Thr), 265 (Gly-->Asp) and 300 (Ser-->Asn). These mutation sites were located in the core region of the lac repressor protein. Temperature-dependent expression of beta-galactosidase was observed in the strains having these mutant lac I genes. By using these temperature-sensitive lac I genes, we developed a thermo-inducible expression system for a foreign gene under the control of the lac promoter. A recombinant fusion protein, consisting of a derivative of E. coli beta-galactosidase and the human calcitonin precursor peptide, was efficiently produced by using this system.
Amino Acid Sequence↗
Control of gene expression in bacteriophage P22 by a small antisense RNA. I. Characterization in vitro of the Psar promoter and the sar RNA transcript.
The characterization in vitro of a newly discovered promoter (Psar) in the bacteriophage P22 immI region is described. Psar is located within the ant gene and is directed toward the major immI promoter, Pant. The entire intercistronic region between the P22 arc and ant genes (69 bp) is transcribed. The initiation and termination of sar (small antisense regulatory) RNA transcription are unusual. Frequent abortive initiation occurs in the presence of all four NTPs; RNA products 3-13 nucleotides in length are produced in about 15- to 25-fold larger numbers than full-length transcripts. Termination of sar RNA synthesis occurs after transcription of the first and second Ts of a TTTA sequence following a region of hyphenated dyad symmetry. The effects of convergent transcription between Pant and Psar were investigated on linear and supercoiled templates. Active transcription from Pant interferes with full-length transcription from Psar; several factors that interfere with Pant initiation (e.g., Pant down-mutation, Mnt repressor protein, Arc repressor protein) result in indirect activation of sar RNA synthesis. The sar RNA pairs rapidly with ant mRNA to form a stable stoichiometric complex. The location and properties of Psar suggest an important regulatory function for sar RNA as a negative effector of ant expression. The results of Wu et al. (this issue) support this suggestion.
Base Sequence↗