Cro repressor protein and its interaction with DNA.
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Bacterial Proteins↗
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Translational control by repressor proteins binding to the 5'UTR of mRNAs.
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5' Untranslated Regions↗
Evidence for leucine zipper motif in lactose repressor protein.
Amino acid sequence homology between the carboxyl-terminal segment of the lac repressor and eukaryotic proteins containing the leucine zipper motif with associated basic DNA binding region (bZIP) has been identified. Based on the sequence comparisons, site-specific mutations have been generated at two sites predicted to participate in oligomer formation based on the three-leucine heptad repeat at positions 342, 349, and 356. Leu342----Ala, Leu349----Ala, and Leu349----Pro have been isolated and their oligomeric state and ligand binding properties evaluated. These mutant proteins do not form tetramers but exist as stable dimers with inducer binding comparable with the wild-type protein. Apparent operator affinities for lac repressor proteins with mutations in the proposed bZIP domain were significantly lower than the corresponding wild-type values. For these dimeric mutant proteins, the monomer-dimer equilibrium is linked to the apparent operator binding constant. The values for the monomer-monomer binding constant and for the intrinsic operator binding constant for the dimer cannot be resolved from measurements of the observed Kd for operator DNA. Further studies on these proteins are in progress.
Amino Acid Sequence↗
Drosophila Topors is a RING finger-containing protein that functions as a ubiquitin-protein isopeptide ligase for the hairy basic helix-loop-helix repressor protein.
Transcriptional repression plays an essential role in many aspects of metazoan development. Drosophila hairy is a primary pair-rule gene encoding a basic helix-loop-helix class transcriptional repressor that is required for proper segmentation. Previous characterization of Hairy-binding proteins has implicated two different classes of histone deacetylase as mediators of Hairy repression. Here, we present the characterization of a novel Hairy-interacting protein (dTopors) that binds specifically to the basic region of Hairy, but does not affect the ability of Hairy to bind DNA. By reducing the gene dose of dtopors, we demonstrate that it acts genetically as an antagonist of Hairy-mediated transcriptional repression. Consistent with this genetic interaction, we show that that recombinant dTopors protein possesses ubiquitin-protein isopeptide ligase activity in vitro and that dTopors mediates Hairy polyubiquitination and can lead to Hairy degradation. This work provides the first evidence that regulated proteolysis of Hairy is required for correct segmentation.
Amino Acid Sequence↗
Translational regulation of the spc operon in Escherichia coli. Identification and structural analysis of the target site for S8 repressor protein.
The spc ribosomal protein operon of Escherichia coli is feedback-regulated by ribosomal protein S8, a translational repressor. We have analyzed the region of the spc mRNA that is responsible for this regulation. First, we have established that the S8 target site on the mRNA is near the translation start site of the third gene encoding ribosomal protein L5 in the operon. This was done by constructing hybrid plasmids carrying spc operon ribosomal protein genes under lac transcriptional control, as well as their deletion derivatives, and carrying out both in vivo and in vitro protein synthesis experiments. Next, the secondary structure of this region was studied by analyzing 5' end-labeled RNA synthesized from the phage SP6 promoter using structure-specific nucleases. A secondary structure model consistent with the results was deduced with the aid of a computer prediction of RNA folding. In addition, we cloned and sequenced the corresponding region from Salmonella typhimurium, Proteus vulgaris and Serratia marcescens and found five "compensating" substitutions that support some of the deduced helical structures of mRNA. None of the base changes was inconsistent with the deduced secondary structure model. Finally, site-directed mutagenesis experiments have identified bases important for regulation, including two base-paired sites representing each of two helical regions. This has led to the conclusion that some specific nucleotide residues located between these two helical regions are directly involved in S8 recognition, and that the function of the two helical regions is to maintain the proper orientation of these nucleotide residues. Comparison of the structure of the S8 target site on the spc mRNA with the known S8 binding site on rRNA has revealed a striking similarity in both primary and secondary structures. In particular, primary sequences of rRNA conserved among distantly related bacterial species in this region is found to be identical with the sequences at the corresponding positions in mRNA. These results suggest that the same structural features of the S8 repressor protein are involved in the interaction with both 16 S rRNA and the mRNA target site.
Base Sequence↗
Functional and structural analysis of HrcA repressor protein from Caulobacter crescentus.
A large number of bacteria regulate chaperone gene expression during heat shock by the HrcA-CIRCE system, in which the DNA element called CIRCE serves as binding site for the repressor protein HrcA under nonstress conditions. In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction is controlled by a sigma32-dependent promoter and the HrcA-CIRCE system plays a role in regulation of groESL expression under physiological temperatures. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein, and specific binding to the CIRCE element was obtained by gel shift assays. The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that the GroE chaperonin machine modulates HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells, producing different amounts of GroES/GroEL. In addition, we identified the putative DNA-binding domain of HrcA through extensive protein sequence comparison and constructed various HrcA mutant proteins containing single amino acid substitutions in or near this region. In vitro and in vivo experiments with these mutated proteins indicated several amino acids important for repressor activity.
Amino Acid Sequence↗
Tyrosine phosphorylation regulates the DNA binding activity of a nuclear factor 1-like repressor protein.
We have previously identified an ubiquitous repressor binding site that binds a nuclear factor 1 (NF-1)-like transcription factor designated BEF-1. The DNA binding activity of BEF-1, a 98-kDa protein, is increased by the oncoproteins of adenovirus, the early region 1a proteins (E1a), which results in the induction of further repression. Using the prototype repressor sequence, first identified in the enhancer of the human polyoma virus BKV-P2 we have shown that phosphorylation of BEF-1 is required for its DNA binding activity. We demonstrate here that the inhibition of DNA binding by BEF-1 dephosphorylated with potato acid phosphatase or calf intestinal alkaline phosphatase was reversed by sodium orthovanadate, a specific inhibitor of phosphotyrosyl-protein phosphatases. In addition, BEF-1 binding activity, but not the binding of related factor NF-1, could be inhibited by dephosphorylation with a specific phosphotyrosine phosphatase. We found that both polyclonal and monoclonal phosphotyrosine-specific antibodies blocked binding of the repressor protein to the BEF-1 site. Moreover, BEF-1 activity could be adsorbed on an anti-phosphotyrosine antibody column and specially eluted with phosphotyrosine. In transfection studies in HeLa cells, which contain high levels of BEF-1, we show that E1a-induced repression mediated by BEF-1 was relieved with the tyrosine kinase inhibitors genistein and tyrphostin. Together, these results demonstrate that a phosphotyrosine on the BEF-1 repressor protein regulates DNA binding activity and thus regulates repression of the BKV-P2 enhancer. This report represents the first demonstration that the phosphorylated state of a tyrosine can control gene expression by altering the DNA binding activity of a transcription factor.
BK Virus↗
Molecular dynamics simulations of the tetracycline-repressor protein: the mechanism of induction.
Molecular dynamics simulations on the tetracycline-repressor (TetR) protein, both in the absence of an inducer and complexed with the inducers tetracycline and 5a,6-anhydrotetracycline, show significant differences in the structures and dynamics of the induced and non-induced forms of the protein. Calpha-density-difference plots, low-frequency normal vibrations and inter-residue interaction energies all point to a common mechanism of induction. The inducer displaces Asp156 from the magnesium ion in the binding pocket, leading to a short cascade of rearrangements of salt bridges that results in the allosteric change. The increased flexibility of the induced form of the protein is suggested to contribute to the decrease in binding affinity to DNA on induction.
Aspartic Acid↗
cAMP-CRP activator complex and the CytR repressor protein bind co-operatively to the cytRP promoter in Escherichia coli and CytR antagonizes the cAMP-CRP-induced DNA bend.
Initiation of transcription from the cytRP promoter in Escherichia coli is activated by the cAMP-CRP complex and negatively regulated by the CytR repressor protein. By combining gel retardation and footprinting assays, we show that cAMP-CRP binds to a single site centered at position -64 and induces a considerable bend in the DNA. CytR binds to a region immediately downstream from, and partially overlapping, the CRP site, and induces a modest bend into the DNA. In combination, cAMP-CRP and CytR bind co-operatively to cytRP forming a nucleoprotein complex in which the proteins directly interact with each other and bind to the same face of the DNA helix. CytR binding concomitantly antagonizes the cAMP-CRP-induced bend. This study indicates that the minimal DNA region required to obtain CytR regulation consists of a single binding site for each of cAMP-CRP and CytR. The case described here, in which a protein-induced DNA bend is modulated by a second protein, may illustrate a mechanism that applies to other regulatory systems.
Bacterial Proteins↗
KRAB-containing zinc-finger repressor proteins.
The largest family of zinc-finger transcription factors comprises those containing the Krüppel-associated box (or KRAB domain), which are present only in tetrapod vertebrates. Many genes encoding KRAB-containing proteins are arranged in clusters in the human genome, with one cluster close to chromosome 9ql3 and others in centromeric and telomeric regions of other chromosomes, but other genes occur individually throughout the genome. The KRAB domain, which is found in the amino-terminal region of the proteins, behaves as a transcriptional repressor domain by binding to corepressor proteins, whereas the C2H2 zinc-finger motifs bind DNA. The functions currently proposed for members of the KRAB-containing protein family include transcriptional repression of RNA polymerase I, II, and III promoters and binding and splicing of RNA. Members of the family are involved in maintenance of the nucleolus, cell differentiation, cell proliferation, apoptosis, and neoplastic transformation.
Amino Acid Motifs↗
Chemical synthesis of a fully active transcriptional repressor protein.
Plasmid pLS1-encoded 45-amino acid transcriptional repressor CopG (formerly RepA) has been chemically synthesized. A one-step purification of the synthetic protein has been developed, which yields high levels of pure protein with low or no contamination of truncated products. We have compared some properties of the chemical CopG protein with those of the biologically purified CopG. The two proteins were indistinguishable in (i) their ability to generate specific protein-DNA complexes, (ii) their capacity to protect a restriction site included within the CopG DNA target, and (iii) in their in vitro capacity to specifically repress synthesis of copG mRNA.
Amino Acid Sequence↗
The function of hairy-related bHLH repressor proteins in cell fate decisions.
Hairy-related proteins are a distinct subfamily of basic helix-loop-helix (bHLH) proteins that generally function as DNA-binding transcriptional repressors. These proteins act in opposition to bHLH transcriptional activator proteins such as the proneural and myogenic proteins; together, the activator and repressor genes that encode these proteins have co-evolved as a regulatory gene "cassette" or "module" for controlling cell fate decisions. In the development of the Drosophila peripheral nervous system, Hairy-related genes function at multiple steps during neurogenesis, for example, as positional information genes that establish the "prepattern" that controls where "proneural cluster" equivalence groups will form, and later as nuclear effectors of the Notch signaling pathway to "single out" individual precursor cells within the equivalence group. Hairy-related genes also function in the establishment and restriction of other types of equivalence groups, such as those for muscle and Malphigian tubule precursors. This general function in cell fate specification has been conserved from Drosophila to vertebrates and has implications for human disease pathogenesis.
Animals↗
The cox protein of bacteriophage P2 inhibits the formation of the repressor protein and autoregulates the early operon.
The cox gene is the first gene of the early operon of bacteriophage P2. The early promoter Pe and the repressor promoter Pc are located close to each other and in such a way that their transcripts have opposite polarity and show an overlap of about 30 nucleotides. The expression of the early operon and of the C gene was studied in vivo by using fusions to a promoterless cat (chloramphenicol acetyl transferase) gene. The results show that the Cox protein negatively autoregulates the early operon and inhibits the formation of the repressor C. By measuring the efficiency of plating of a series of P2 virulent deletion mutants on bacteria carrying a cloned cox gene, the site of action of the Cox protein was mapped within the Pe-Pc region. The stimulatory effect of the C protein on expression of the Pc promoter was found to be due to inhibition of transcription from Pc; this was demonstrated by mutating Pe which showed that loss of transcription from Pe stimulated transcription from Pc. Hence, this is a case of regulation of gene expression by convergent transcription. By cloning the region C-Pe-Pc-cox such that the cat and kan genes are expressed from Pc and Pe, respectively, it was shown that only one of the resistances (Cm and Km) was expressed. This mimics the choice between lysogeny and lytic growth of the phage. The 'lysogenic' state was very stable whereas the 'lytic' state flipped to the 'lysogenic' at a somewhat higher frequency. The presence of a cloned cox gene drastically stimulated the formation of free phage from a P2-lysogen and dramatically reduced the frequency of lysogenization after P2 infection. We conclude that the pleiotropic effects of the cox (control of excision) gene, namely effects on lysogenization, formation of free phage and site-specific P2 recombination, can be explained by the effect of the Cox protein on the activity of the promoters Pc and Pe.
Amino Acid Sequence↗
Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the pMV158-encoded plasmid transcriptional repressor protein CopG.
Plasmid pMV158 encodes a 45 amino acid transcriptional repressor, CopG, which is involved in copy number control. A new procedure for overproduction and purification of the protein has been developed. The CopG protein thus obtained retained its ability to specifically bind to DNA and to repress its own promoter. Purified CopG protein has been crystallized using the sitting-drop vapor diffusion method. The crystals, belonging to orthorhombic space group C222(1) (cell constants a = 67.2 A, b = 102.5 A, c = 40.2 A), were obtained from a solution containing methylpentanediol, benzamidine and sodium chloride, buffered to pH 6.7. Complete diffraction data up to 1.6 A resolution have been collected. Considerations about the Matthews parameter account for the most likely presence of three molecules in the asymmetric unit (2.27 A3/Da).
Amino Acid Sequence↗
Expression of the transcriptional repressor protein Kid-1 leads to the disintegration of the nucleolus.
The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two Cys2His2-zinc finger clusters of four and nine zinc fingers at the COOH terminus. It was the first KRAB-zinc finger protein for which a transcriptional repressor activity was demonstrated. Subsequently, the KRAB-A domain was identified as a widespread transcriptional repressor motif. We now present a biochemical and functional analysis of the Kid-1 protein in transfected cells. The full-length Kid-1 protein is targeted to the nucleolus and adheres tightly to as yet undefined nucleolar structures, leading eventually to the disintegration of the nucleolus. The tight adherence and nucleolar distribution can be attributed to the larger zinc finger cluster, whereas the KRAB-A domain is responsible for the nucleolar fragmentation. Upon disintegration of the nucleolus, the nucleolar transcription factor upstream binding factor disappears from the nucleolar fragments. In the absence of Kid-1, the KRIP-1 protein, which represents the natural interacting partner of zinc finger proteins with a KRAB-A domain, is homogeneously distributed in the nucleus, whereas coexpression of Kid-1 leads to a shift of KRIP-1 into the nucleolus. Nucleolar run-ons demonstrate that rDNA transcription is shut off in the nucleolar fragments. Our data demonstrate the functional diversity of the KRAB and zinc finger domains of Kid-1 and provide new functional insights into the regulation of the nucleolar structure.
Animals↗
Protein-DNA interaction-based detection of small molecules by employing Renilla luciferase fusion protein: quantitative and generic measurement of tetracyclines with a Renilla luciferase-tagged Tet repressor protein.
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Base Sequence↗
The transcriptional repressor protein PRH interacts with the proteasome.
PRH (proline-rich homeodomain protein)/Hex is important in the control of cell proliferation and differentiation. We have shown previously that PRH contains two domains that can bring about transcriptional repression independently; the PRH homeodomain represses transcription by binding to TATA box sequences, whereas the proline-rich N-terminal domain can repress transcription by interacting with members of the Groucho/TLE (transducin-like enhancer of split) family of co-repressor proteins. The proteasome is a multi-subunit protein complex involved in the processing and degradation of proteins. Some proteasome subunits have been suggested to play a role in the regulation of transcription. In the present study, we show that PRH interacts with the HC8 subunit of the proteasome in the context of both 20 and 26 S proteasomes. Moreover, we show that PRH is associated with the proteasome in haematopoietic cells and that the proline-rich PRH N-terminal domain is responsible for this interaction. Whereas PRH can be cleaved by the proteasome, it does not appear to be degraded rapidly in vitro or in vivo, and the proteolytic activity of the proteasome is not required for transcriptional repression by PRH. However, proteasomal digestion of PRH can liberate truncated PRH proteins that retain the ability to bind to DNA. We discuss these findings in terms of the biological role of PRH in gene regulation and the control of cell proliferation.
Antibodies, Monoclonal↗
High resolution structural studies of Cro repressor protein and implications for DNA recognition.
Cro repressor is a small dimeric protein that binds to specific sites on the DNA of bacteriophage lambda. The structure of Cro has been determined and suggests that the protein binds to its sequence-specific sites with a pair of two-fold related alpha-helices of the protein located within successive major grooves of the DNA. From the known three-dimensional structure of the repressor, model building and energy refinement have been used to develop a detailed model for the presumed complex between Cro and DNA. Recognition of specific DNA binding sites appears to occur via multiple hydrogen bonds between amino acid side chains of the protein and base pair atoms exposed within the major groove of DNA. The Cro:DNA model is consistent with the calculated electrostatic potential energy surface of the protein. From a series of amino acid sequence and gene sequence comparisons, it appears that a number of other DNA-binding proteins have an alpha-helical DNA-binding region similar to that seen in Cro. The apparent sequence homology includes not only DNA-binding proteins from different bacteriophages, but also gene-regulatory proteins from bacteria and yeast. It has also been found that the conformations of part of the presumed DNA-binding regions of Cro repressor, lambda repressor and CAP gene activator proteins are strikingly similar. Taken together, these results strongly suggest that a two-helical structural unit occurs in the DNA-binding region of many proteins that regulate gene expression. However, the results to date do not suggest that there is a simple one-to-one recognition code between amino acids and bases. Crystals have been obtained of complexes of Cro with six-base-pair and nine-basepair DNA oligomers, and X-ray analysis of these co-crystals is in progress.
Amino Acid Sequence↗