Differential protein binding in lymphocytes to a sequence in the enhancer of the mouse retrovirus SL3-3.

An electrophoretic mobility shift assay was used to characterize interactions of nuclear proteins with a DNA segment in the enhancer element of the leukemogenic murine retrovirus SL3-3. Mutation of a DNA sequence of the 5'-TGTGG-3' type decreased transcription in vivo specifically in T-lymphocyte cell lines. Extracts of nuclei from different T-lymphocyte cell lines or cells from lymphoid organs resulted in much higher amounts of complexes in vitro with this DNA sequence than did extracts from other cell lines or organs tested. Differences were also found in the sets of complexes obtained with extracts from the different types of cells. The DNA sequence specificities of the different SL3-3 enhancer factor 1 (SEF1) protein complexes were found to be distinct from those of several other previously identified DNA motifs of the TGTGG type because of differences in several nucleotides critical for binding and because these other DNA motifs could not compete with the identified DNA sequence for binding of SEF1. Limited treatment with several different proteases cleaved the SEF1 proteins such that their DNA-binding domain(s) remained and created complexes with decreased and nondistinguishable electrophoretic mobility shifts and with new properties. These results indicate that the SEF1 proteins have a structure with a flexible and relatively vulnerable hinge region linking a DNA-binding domain(s) to a more variable domain(s) with other functions. We suggest that the binding of SEF1 is an essential factor for the T-cell tropism of SL3-3 and the ability of this virus to cause T-cell lymphomas.

Specific protein binding to the simian virus 40 enhancer in vitro.

HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains.

Calcium regulation of basic helix-loop-helix transcription factors.

The basic helix-loop-helix (bHLH) family of transcription factors is essential for numerous developmental and growth control processes. The regulation of bHLH proteins occurs at many levels, including tissue specific expression, differential oligomerization and DNA binding specificities, interaction with negatively acting HLH proteins and post-translational modifications. This review focuses on what is emerging as another level of bHLH protein regulation, calcium regulation through interaction with Ca2+ loaded calmodulin and S-100 proteins. The mechanism and implications of these Ca2+ regulated interactions are discussed.

A novel target recognition revealed by calmodulin in complex with the basic helix--loop--helix transcription factor SEF2-1/E2-2.

Calmodulin is the predominant intracellular receptor for Ca(2+) signals, mediating the regulation of numerous cellular processes. It can inhibit the DNA binding of basic helix--loop--helix transcription factors by a direct interaction of a novel type. To structurally characterize this novel calmodulin-target interaction, we decided to study the complex of calmodulin with a dimeric peptide corresponding to the DNA-binding domains of the dimeric basic helix-loop-helix transcription factor SEF2-1 (SEF2-1mp) using NMR. Here, we report that the stoichiometry of the calmodulin:SEF2-1mp complex is one dimeric peptide binding two calmodulin molecules. We also report the 1H, 13C, and 15N resonance assignments and the secondary structure of calmodulin in this for NMR large (approximately 38 kD) complex, as well as the 1H assignments and secondary structure of SEF2-1mp. In addition, we determined the amide proton exchange rates of calmodulin and measured intermolecular calmodulin:SEF2-1mp and calmodulin:calmodulin NOE contacts. The isotope-filtered experiments show a large number of SEF2-1mp to calmodulin NOE contacts indicating that a tight complex is formed, which is confirmed by an intermolecular calmodulin:calmodulin NOE contact. The secondary structure and amide proton exchange data show that the binding does not occur via the classical wraparound binding mode. Instead, the data indicate that calmodulin interacts with SEF2-1mp in a more open conformation, although the hydrophobic surfaces of the N- and C-terminal domains still form the main interaction sites. Interactions involving charged residues are also identified in agreement with the known relatively high sensitivity of the binding to ionic strength. Finally, the peptide does not form an alpha-helix as in the classical wraparound binding mode.

Nucleotide and calcium-induced conformational changes in histone H1.

The principal constituents of chromatin, histone H1 (H1) and the nucleosome have essential roles in regulation of eukaryotic gene expression. However, mechanisms for the H1-dependent inactivation and for the ATP-dependent chromatin remodeling upon activation are largely unelucidated. Using circular dichroism (CD) analysis we show that ATP and other nucleotides and Ca2+ induce structural changes in H1. ATP and Ca2+ also induce changes when H1 is interacting with DNA, and the changes in H1 are accompanied by alterations in its DNA interaction. These results suggest that nucleotide and Ca2+ binding may be important for H1-mediated chromatin changes.

Calmodulin dependence of NFkappaB activation.

The NFkappaB family of transcription factors is regulated by inhibitory IkappaB proteins. A diversity of stimuli leads to the phosphorylation and subsequent degradation of IkappaB, releasing NFkappaB to act on its target genes. Calmodulin (CaM) is a key regulator of numerous cellular processes and is the predominant intracellular receptor for Ca2+ signals. Here we report that several CaM antagonists inhibit the activation of NFkappaB, and that this is due to the prevention of inducible IkappaB phosphorylation. Our results suggest that CaM is involved in the phosphorylation of IkappaB, a finding that may help in elucidating the mechanism of this critical step of NFkappaB activation.

DNA binding of histone H1 is modulated by nucleotides.

Histone H1 acts as a general repressor of transcription in eukaryotes by organizing nucleosomes into inaccessible condensed forms of chromatin. The capability of H1 to bind to DNA with some sequence specificity is likely to be critical in the control of these processes. We show here that ATP and several other nucleotides, including non-hydrolyzable derivatives, can inhibit DNA binding of H1. The results also show that ATP differentially affects binding of H1 to DNA in a fashion enhancing nucleotide sequence specificity of the binding. The study suggests a novel mechanism of modulation of H1 activity that has important implications for the role of H1 as a transcriptional regulator.

Chloride binding by the AML1/Runx1 transcription factor studied by NMR.

It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.

Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion.

Two different lengths of the gene encoding Escherichia coli heat-stable toxin (STa) were fused to the carboxy end of the gene coding for the E. coli heat-labile toxin A-subunit (LTA). The hybrid genes directed expression of chimeric LTA-STa proteins. Association of these chimeras with native heat-labile toxin B-subunit (LTB) resulted in protein complexes that bound to GM1 ganglioside and thereby could be assayed in a GM1 ELISA. The complexes reacted with monoclonal antibodies against either LTA, LTB or STa indicating that the STa and LT epitopes remained immunologically intact after fusion. Genetically constructed chimeric proteins exhibiting LT and STa antigens on the same molecule may represent a promising approach to development of broadly protective immunoprophylactic agents and/or useful immunodiagnostic reagents for diarrhoeal diseases caused by enterotoxinogenic E. coli.

Initiation of translation makes attenuation of ampC in E. coli dependent on growth rate.

The chromosomal beta-lactamase gene of E. coli, ampC, shows increased expression with increased growth rate of the bacteria. We have previously shown that transcription of ampC is attenuated, and that a mutation in the terminator stem of this attenuator abolishes the growth rate-dependency of ampC expression. We now present studies using mutations, made in vitro, located such that the 5'-end of ampC mRNA carries a possible recognition sequence for initiation of translation close to the attenuator stem. Alteration of the supposed initiation codon AUG to UUG resulted in a reduced and growth rate-independent expression of ampC beta-lactamase. AmpC mRNA starts with the sequence AUC, which might be a non-typical ribosome binding site, situated four bases before the AUG. Deletion of the C in this sequence caused a partial reduction of ampC expression and also a partial loss of the growth rate-dependent regulation. The phenotypes of these mutants support a model in which formation of a ribosome initiation complex at a level increasing with the growth rate inhibits termination of transcription at the ampC attenuator.

Tissue specific sequence motifs in the enhancer of the leukaemogenic mouse retrovirus SL3-3.

The long terminal repeat (LTR) of the retrovirus SL3-3 determines its tropism for T-lymphocytes and its ability to induce T-cell lymphomas in mice. We have studied the ability of different DNA sequences located upstream of the "TATA" box in the LTR of SL3-3 to enhance transcription in T-lymphocyte cell lines and other cell lines, employing a transient assay and quantitative S1 nuclease mapping. The enhancer was found to be composed of many DNA domains which determines different activities in different cell lines. We find enhancer sequence motifs with a high T-lymphocyte specificity in the DNA repetitions of the LTR, and other enhancer motifs active in a broader range of cells in the surrounding DNA segments. The localization of sequences preferentially active in T-cells within the repeated sequences containing differences between SL3-3 and the very closely related Akv virus, which is without the T cell tropism and leukaemogenicity of SL3-3, supports the notion that the enhancer sequence motifs with T-cell preferences are primary determinants of these properties.

Overlap between ampC and frd operons on the Escherichia coli chromosome.

The promoter for the Escherichia coli ampC beta-lactamase gene is shown to be located within the last gene of the fumarate reductase (frd) operon. Evidence is presented that the ampC attenuator serves as the terminator for transcription of this preceding operon. The nucleotide sequence was determined for two proteins that were shown to be encoded by a DNA segment preceding the ampC gene. The two proteins consisted of 131 and 119 triplets and had molecular weights of 15,000 and 13,100, respectively. The twelve COOH-terminal amino acids of the 13,100 molecular weight protein were found to overlap the ampC promoter. Accordingly, a G.C insertion in the promoter gave both increased transcription of ampC and a frameshift in this overlapping gene, resulting in readthrough proteins. Thus, we describe a type of very compact genetic organization of operons in prokaryotes.

ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of beta-lactamases of the penicillinase type.

A 1536-nucleotide-long sequence that carries the ampC beta-lactamase gene of the Escherichia coli K-12 chromosome has been determined. This gene codes for a protein of 377 amino acids, of which the first 19 amino acids form a signal peptide. The molecular weight of the mature enzyme was determined to be 39,600. The ampC beta-lactamase with a substrate specificity for cephalosporins showed no significant sequence homologies with beta-lactamases of the penicillinase type or with D-alanine carboxypeptidases. However, because the region around serine-80 of the ampC beta-lactamase has extensive homology with an active-site fragment of the Pseudomonas aeruginosa cephalosporinase, we suggest that the ampC cephalosporinase as well as related cephalosporinases form a distinct group of serine beta-lactamases that have an evolutionary origin different from that of the serine penicillinases and thus constitute a new class of beta-lactamases.

Binding of SL3-3 enhancer factor 1 transcriptional activators to viral and chromosomal enhancer sequences.

Interactions between SL3-3 enhancer factor 1 (SEF1) proteins and the enhancer of the murine leukemia virus SL3-3 were analyzed. SEF1 proteins were found to interact with two different DNA sequences within the DNA repeat region of the enhancer; these two motifs cooperated in enhancing initiation of transcription in T lymphocytes. Using an electrophoretic mobility shift assay, we identified nucleotides that are important for the SEF1 binding, and we deduced a sequence, 5'-TTTGCGGTTA/T-3' with highly improved binding of SEF1 proteins. We show that many different SEF1 binding sequences exist in the transcription control regions of different viral and cellular genes. The results indicate a general role of SEF1 proteins in T-cell gene expression.

Basic helix-loop-helix protein sequences determining differential inhibition by calmodulin and S-100 proteins.

Basic helix-loop-helix (bHLH) proteins are a group of transcription factors that are involved in differentiation and numerous other cellular processes. The proteins include the widely expressed class A bHLH proteins (E proteins) and the tissue-specific class B proteins. Previous studies have shown that calmodulin can inhibit the DNA binding activity of certain E proteins but not their heterodimers with class B proteins. Here we show that calmodulin binds to the DNA-interacting basic sequence within the bHLH domain of E proteins. The strength of the binding of bHLH proteins to calmodulin correlates directly with the calmodulin sensitivity of their DNA binding. The basic sequence of MyoD, a class B protein, can also interact with calmodulin. This interaction, however, is blocked by MyoD sequences directly N-terminal of the basic sequence. We further demonstrate that S-100 proteins can interact with and differentially inhibit the DNA binding of bHLH proteins through interaction with the basic sequence. Both the binding to the basic sequence and the effect of the directly N-terminal sequence vary for different S-100 proteins and bHLH proteins. The results suggest the involvement of both calmodulin and S-100 proteins in the differential regulation of bHLH proteins.

Isolation and characterization of DNA repetitions carrying the chromosomal beta-lactamase gene of Escherichia coli K-12.

A ColEl hybrid plasmid, pNUl, carrying the amp operon coding for chromosomal beta-lactamase was isolated from the Clarke and Carbon collection and physically mapped. The physical location of ampC within this plasmid was further deduced by in vitro cloning. By reciprocal recombination between pNUl and chromosome of two unstable beta-lactamase hyperproducing E. coli K-12 mutants a large plasmid from each mutant was obtained. The respective plasmid was physically mapped and found to contain five and two repeated DNA segments. The repetitions within each plasmid were equal in size, 9,800 bp and 11,900 bp respectively and were organized in tandem. The end points of the repeats were different in the two plasmids but shared a DNA segment carrying the ampC gene. The chromosomal DNA of the beta-lactamase hyperproducing E. coli mutants were found to contain an amplified DNA segment equal in size to the repeated unit found in the respective plasmid. The data shows that up to 10 identical repeats organized in tandem can be generated by a normal mutation frequency in E. coli.

A novel type of calmodulin interaction in the inhibition of basic helix-loop-helix transcription factors.

Calmodulin is the predominant intracellular receptor for Ca(2+) signals, mediating the regulation of numerous cellular processes. Previous studies have shown that calcium-loaded calmodulin can bind to and inhibit the activity of certain basic helix-loop-helix (bHLH) transcription factors. The basic sequence within the bHLH domain is the primary target for calmodulin binding, and sequences modulating the calmodulin interaction reside directly N-terminal to the basic sequence. Here we show that the interaction of calmodulin with bHLH proteins is of a novel type, displaying characteristics very different from those of previously characterized calmodulin-target complexes. We show that calmodulin interacts much stronger with a dimeric basic sequence than with the monomeric form. The calmodulin-bHLH protein complex contains equimolar amounts of calmodulin and bHLH chains. The interaction is unusual in being to a large extent polar in nature, and it is highly resistant to tested calmodulin inhibitors. Both the N-terminal and C-terminal domains of calmodulin can independently bind to and inhibit the DNA binding of bHLH proteins. The C-terminal domain preferentially binds to the basic sequence, whereas the N-terminal domain is essential for the effect of the modulatory sequence. We propose a model for the calmodulin-bHLH complex where two calmodulin molecules interact with one bHLH dimer, with one domain of calmodulin preferentially binding to the basic sequence of bHLH proteins and the other domain interacting with the modulatory sequence.

Purification of SEF1 proteins binding to transcriptional enhancer elements active in T lymphocytes.

Binding sites for SL3-3 enhancer factor 1 (SEF1) are important for the transcriptional activity in T lymphocytes and the tumorigenicity of SL3-3 murine leukemia virus. SEF1 is also implicated in the activity of many other leukemia, lymphoma, and sarcoma virus enhancers, and enhancers of genes for T cell receptor-CD3 subunits. We have purified several proteins binding to SEF1 sites from bovine thymus using a five-step purification procedure. The proteins migrated as 19 distinct bands representing molecular masses from 23 kDa to about 200 kDa in SDS-polyacrylamide gel electrophoresis. Ten DNA binding proteins, with molecular masses between 23 and 67 kDa, could be isolated after separation by SDS-polyacrylamide gel electrophoresis. The DNA binding specificities of these proteins were similar and corresponded to that of the SEF1 binding activity in nuclear extracts. Each of these isolated SEF1 proteins also bound to the essential delta-E3 element of the human T cell receptor delta enhancer. Antibodies against one SEF1 protein only reacted with the protein used for immunization, which indicates a limited homology between at least some SEF1 proteins. We also present data suggesting that SEF1 proteins exist in multiple forms with differences in their DNA binding specificity, and that high affinity DNA binding of the SEF1 proteins requires protein phosphorylation.

Sequence elements determining ampC promoter strength in E. coli.

A number of spontaneous up-promoter mutations have been isolated in the ampC beta-lactamase gene of Escherichia coli. The mutants were analyzed by DNA sequencing, and the level of ampC gene expression was determined. Six mutants with a 21-fold increase in promoter strength compared with the wild-type were mutated in the -35 promoter region from TTGTCA to the consensus sequence TTGACA . The -10 region sequence TACAAT was mutated to the consensus sequence TATAAT in three mutants exhibiting an ampC promoter seven times stronger than the wild-type. We have previously described a 1-bp insertion mutant ( Jaurin et al., 1981) that changes the inter-region distance to the consensus 17 bp. Thus, all the up-mutations found in the ampC promoter represent corrections of the three major discrepancies between the ampC promoter and the consensus E. coli promoter. We conclude that the three consensus elements of E. coli promoters, the -35 and -10 regions and an optimal inter-region distance of 17 bp, are the main elements determining the promoter strength.