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PubMed1996

The coupling between ftsZ transcription and initiation of DNA replication is not mediated by the DnaA-boxes upstream of ftsZ or by DnaA.

The DnaA protein of Escherichia coli is a multi-functional protein which, In addition to promoting initiation of replication, can regulate the initiation or termination of transcription of a variety of genes. It acts by binding to DNA at a defined sequence, termed a DnaA-box. Three candidate DnaA-boxes which occur within the essential cell-division genes, ftsQ and ftsA, have been hypothesized to mediate the response of the downstream ftsZ gene to intracellular levels of DnaA, and thus to couple the processes of initiation and cell division. We show here that, although transcription from promoters upstream of ftsZ is increased when initiation of chromosome replication is blocked by DnaA inactivation, this response is not mediated by the DnaA-boxes near these promoters, nor is it specific to DnaA. We show, furthermore, that mutational inactivation of the putative DnaA-binding sites in the fts region of the chromosome does not lead to impaired growth or reduced survival of cells.

Bacterial Proteins↗

PubMed2000

Recruitment of the yeast MADS-box proteins, ArgRI and Mcm1 by the pleiotropic factor ArgRIII is required for their stability.

Regulation of arginine metabolism requires the integrity of four regulatory proteins, ArgRI, ArgRII, ArgRIII and Mcm1. To characterize further the interactions between the different proteins, we used the two-hybrid system, which showed that ArgRI and Mcm1 interact together, and with ArgRII and ArgRIII, without an arginine requirement. To define the interacting domains of ArgRI and Mcm1 with ArgRIII, we fused portions of ArgRI and Mcm1 to the DNA-binding domain of Gal4 (GBD) and created mutations in GBD-ArgRI and GBD-Mcm1. The putative alpha helix present in the MADS-box domain of ArgRI and Mcm1 is their major region of interaction with ArgRIII. Interactions between the two MADS-box proteins and ArgRIII were confirmed using affinity chromatography. The requirement for ArgRIII in the control of arginine metabolism can be bypassed in vitro as well as in vivo by overproducing ArgRI or Mcm1, which indicates that ArgRIII is not present in the protein complex formed with the 'arginine boxes'. We show that the impairment of arginine regulation in an argRIII deletant strain is a result of a lack of stability of ArgRI and Mcm1. A mutation in ArgRI, impairing its interaction with ArgRIII, leads to an unstable ArgRI protein in a wild-type strain. ArgRIII integrity is crucial for Mcm1 function, as shown by the marked decreased expression of five genes controlled by Mcm1. However, ArgRIII is likely to recruit other proteins in the yeast cell, as overexpression of Mcm1 does not compensate some physiological defects observed in an argRIII deletant strain.

Amino Acid Sequence↗

PubMed2003

The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis.

The subtilin gene cluster (spa) of Bacillus subtilis ATCC 6633 is organized in transcriptional units spaBTC, spaS, spaIFEG and spaRK. Specific binding of the response regulator protein SpaR to spaB, spaS and spaI DNA promoter fragments was shown by means of electromobility shift assays. A repeated pentanucleotide sequence spaced by six nucleotides was identified as SpaR binding motif (spa-box). Saturating mutational analysis of the spa-box by single- and multiple-base-pair substitutions revealed the consensus motif (A/T)TGAT for optimal SpaR binding with the second, third and fifth position being absolutely conservative. Variations in the spacer size between the two pentanucleotide repeats revealed a strong conservation of their relative location. Only DNA with a proximal arrangement of two pentanucleotide repeats showed affinity to SpaR. A 2:1 stoichiometry between SpaR and DNA was obtained by optical biosensor analyses, which corresponds to the binding of two SpaR proteins per spa-box.

Amino Acid Sequence↗

PubMed1995

A characterization of the MADS-box gene family in maize.

Studies on distantly related dicot plant species have identified homeotic genes that specify floral meristem identity and determine the fate of floral organ primordia. Most of these genes belong to a family characterized by the presence of a structural motif, the MADS-box, which encodes a protein domain with DNA-binding properties. As part of an effort to understand how such genes may have been recruited during the evolution of flowers with different organ types such as those found in maize, two members of this gene family in maize, ZAG1 and ZAG2, have been characterized previously. Here, the isolation and characterization of four new members of this gene family, designated ZAP1, ZAG3, ZAG4 and ZAG5, are described and the genetic map position of these and 28 additional maize MADS-box genes is determined. The first new member of this family appears to be the Zea mays ortholog of the floral homeotic gene APETALA1 (AP1) and has been designated ZAP1. One of these genes, ZAG4, is unusual in that its deduced protein sequence includes the MADS domain but lacks the K-domain characteristically present in this family of genes. In addition, its copy number and expression varies among different inbreds. A large number of maize MADS-box genes map to duplicated regions of the genome, including one pair characterized here, ZAG3 and ZAG5. These data underscore the complexity of this gene family in maize, and provide the basis for further studies into the regulation of floral organ morphogenesis among the grasses.

Amino Acid Sequence↗

PubMed1999

The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required for normal patterning and growth in the floral meristem.

Genetic and molecular studies have suggested that the UNUSUAL FLORAL ORGANS (UFO) gene, from Arabidopsis thaliana, is expressed in all shoot apical meristems, and is involved in the regulation of a complex set of developmental events during floral development, including floral meristem and floral organ identity. Results from in situ hybridization using genes expressed early in floral development as probes indicate that UFO controls growth of young floral primordia. Transgenic constructs were used to provide evidence that UFO regulates floral organ identity by activating or maintaining transcription of the class B organ-identity gene APETALA 3, but not PISTILLATA. In an attempt to understand the biochemical mode of action of the UFO gene product, we show here that UFO is an F-box protein that interacts with Arabidopsis SKP1-like proteins, both in the yeast two-hybrid system and in vitro. In yeast and other organisms both F-box proteins and SKP1 homologues are subunits of specific ubiquitin E3 enzyme complexes that target specific proteins for degradation. The protein selected for degradation by the complex is specified by the F-box proteins. It is therefore possible that the role of UFO is to target for degradation specific proteins controlling normal growth patterns in the floral primordia, as well as proteins that negatively regulate APETALA 3 transcription.

Amino Acid Sequence↗

PubMed2000

Quantitative nature of the Prolamin-box, ACGT and AACA motifs in a rice glutelin gene promoter: minimal cis-element requirements for endosperm-specific gene expression.

The -197 bp promoter of the rice seed storage protein gene, GluB-1, is capable of conferring endosperm-specific gene expression. This proximal 5' flanking region contains four motifs, GCN4, AACA, ACGT and Prolamin-box, which are conserved in many seed storage protein genes. We previously showed that multiple copies of GCN4 conferred endosperm expression pattern when fused to the -46 core promoter of CaMV 35S. In this paper we demonstrate, using a similar approach, that tandem repeated copies of any of the other three motifs are unable to direct expression in seeds as well as other tissues of transgenic rice plants. Mutational analysis of individual motifs in the -197 bp promoter resulted in remarkable reductions in promoter activity. These results indicate that the GCN4 motif acts as an essential element determining endosperm-specific expression and that the AACA, ACGT and Prolamin-box are involved in quantitative regulation of the GluB-1 gene. A set of gain-of-function experiments using transgenic rice showed that either the Prolamin-box or AACA, although often coupled with GCN4 in many genes, is insufficient to form a functional promoter unit with GCN4, whereas a combination of GCN4, AACA and ACGT motifs was found sufficient to confer a detectable level of endosperm expression. Taken together, our results provide direct insight into the importance of combinatorial interplay between cis-elements in regulating the expression of seed storage protein genes.

Base Sequence↗

PubMed2001

The MADS-box gene DEFH28 from Antirrhinum is involved in the regulation of floral meristem identity and fruit development.

DEFH28 is a novel MADS-box gene from Antirrhinum majus. Phylogenetic reconstruction indicates that it belongs to the SQUA-subfamily of MADS-box genes. Based on its expression pattern and the phenotype of transgenic plants it is predicted that DEFH28 exerts a dual function during flower development, namely control of meristem identity and fruit development. Firstly, DEFH28 is expressed in the inflorescence apical meristem and might control, together with SQUAMOSA (SQUA), floral meristem identity in Antirrhinum. Also, DEFH28 is sufficient to switch inflorescence shoot meristem to a floral fate in transgenic Arabidopsis thaliana plants. Secondly, DEFH28 is expressed in carpel walls, where it may regulate carpel wall differentiation and fruit maturation. Support for this later role comes from overexpression of DEFH28 throughout the silique in transgenic Arabidopsis plants where it altered the identity of the replum and valve margin cells so that they adopted a valve cell identity. This late aspect of the DEFH28 function is identical to the FRUITFULL (FUL) function of Arabidopsis as demonstrated in gain-of-function plants. FUL, like DEFH28, belongs to the SQUA-subfamily of MADS-box genes. DEFH28 most likely represents the ortholog of FUL. Promoter analysis shows that the control mechanism conferring a carpel wall specific expression has been conserved between Antirrhinum and Arabidopsis during evolution. Although the overall flower development between Antirrhinum and Arabidopsis is very similar, their carpels mature into different types of fruits: capsules and siliques, respectively. Therefore, it is suggested that the role of DEFH28 in control of carpel wall differentiation reflects a conserved molecular mechanism integrated into two very different carpel developmental pathways.

Amino Acid Sequence↗

PubMed2001

Two B or not two B? Overview of the rapidly expanding B-box family of proteins.

The B-box gene family represents a large number of genes involved in functions such as axial patterning, growth control, differentiation, and transcriptional regulation. These genes possess several conserved motifs that always include a B-box zinc binding motif associated with various other motifs such as the RING zinc finger, an alpha-helical coiled-coil, the rfp or B30.2 motif, propeller domain, and the NHL motif in various combinations. Mutations or rearrangements in several B-box family members are associated with human diseases and cancers such as familial Mediterranean fever (FMF), Optiz/BBB syndrome, acute promyelocytic leukemia, mulibrey nanism, and thyroid carcinomas. This suggests that members of this gene family play important roles in fundamental biological processes. Here we discuss the known members of this rapidly expanding protein family.

Amino Acid Motifs↗

PubMed2002

Mutations within the conserved MADS box of the D-MEF2 muscle differentiation factor result in a loss of DNA binding ability and lethality in Drosophila.

Members of the myocyte enhancer factor 2 (MEF2) family of transcription factors contain highly conserved sequences within their MADS box and MEF2 domain. These motifs are required for DNA binding and dimerization properties, as well as for MEF2 association with various transcriptional activator or repressor proteins. The D-mef2 gene encodes the MEF2 protein of Drosophila and genetic studies have shown that normal D-MEF2 function is needed for muscle cell differentiation during embryogenesis and indirect flight muscle formation during pupal development. We have characterized three additional lethal alleles of D-mef2 and identified the specific mutation in each that alters a conserved amino acid present within the MADS box of all known MEF2 proteins. Mutation of these invariant residues results in the inability of mutant D-MEF2 proteins to bind DNA in vitro, muscle defects within the embryo, and adverse effects on the structure of indirect flight muscles within the adult. Since the crystal structure of a MEF2 core protein bound to DNA has been previously solved, our results correlate the mutation of specific MADS box amino acids utilized for target DNA recognition with severe myogenic phenotypes manifested during Drosophila development.

Amino Acid Sequence↗

PubMed1998

Formation of large nucleoprotein complexes upon binding of the high-mobility-group (HMG) box B-domain of HMG1 protein to supercoiled DNA.

High-mobility group (HMG) 1 is a relatively highly abundant chromosomal protein with structural- rather than sequence-specific preference for binding to DNA. HMG1 has two highly related, folded domains A and B (HMG boxes), attached by a short basic region to an acidic C-terminal domain. We have studied binding of the B-domain of HMG1 protein and its mutants to supercoiled DNA by a gel-retardation assay and electron microscopy. Using a gel-retardation assay, we have demonstrated that HMG1 or HMG1 lacking the acidic C-terminal domain [i.e. HMG1(A+B) bi-domain], but not the isolated B-domain, could preferentially bind supercoiled over-relaxed closed circular or linear DNA. Mutational analysis of the HMG1 B-domain revealed that replacement of Lys96 of the extended N-terminal segment (and much less the neighboring Arg97) and Lys128 of helix II to glutamic acid severely impaired binding of the HMG box domain to supercoiled DNA. The latter mutation within helix II significantly decreased the alpha-helical content of the B-domain as revealed by circular dichroism. We have also shown that mutation of several residues within helix I of the B-domain, in particular Arg110, resulted in a diminished binding to supercoiled DNA as revealed by intensive smearing and reduced retardation of the protein/DNA complexes. These findings indicated that the extended N-terminus, helix I and helix II of the HMG1 B-domain are likely in contact with DNA. Electron microscopy revealed that the B-domain could bind to supercoiled DNA at higher HMG/DNA molar ratios as oligomeric protein beads with subsequent association of the beads into large nucleoprotein complexes from which many looped DNA molecules emerged. Most of the introduced mutations within all three helices of the B-domain (involving mainly basic and aromatic residues) abolished formation of the large nucleoprotein complexes even though the binding of the HMG box to supercoiled DNA was retained as revealed by a gel-retardation assay. A model for the interaction of the B-domain of HMG 1 with supercoiled DNA is presented and discussed.

Amino Acid Sequence↗

PubMed2000

Cdc20 protein contains a destruction-box but, unlike Clb2, its proteolysisis not acutely dependent on the activity of anaphase-promoting complex.

Both chromosome segregation and the final exit from mitosis require a ubiquitin-protein ligase called anaphase-promoting complex (APC) or cyclosome. This multiprotein complex ubiquitinates various substrates, such as the anaphase inhibitor Pds1 and mitotic cyclins, and thus targets them for proteolysis by the 26S proteasome. The ubiquitination by APC is dependent on the presence of a destruction-box sequence in the N-terminus of target proteins. Recent reports have strongly suggested that Cdc20, a WD40 repeat-containing protein required for nuclear division in the budding yeast Saccharomyces cerevisiae, is essential for the APC-mediated proteolysis. To understand the function of CDC20, we have studied its regulation in some detail. The expression of the CDC20 gene is cell-cycle regulated such that it is transcribed only during late S phase and mitosis. Although the protein is unstable to some extent through out the cell cycle, its degradation is particularly enhanced in G1. Cdc20 contains a destruction box sequence which, when mutated or deleted, stabilizes it considerably in G1. Surprisingly, we find that while the inactivation of APC subunits Cdc16, Cdc23 or Cdc27 results in stabilization of the mitotic cyclin Clb2 in G1, the proteolytic destruction of Cdc20 remains largely unaffected. This suggests the existence of proteolytic mechanisms in G1 that can degrade destruction-box containing proteins, such as Cdc20, in an APC-independent manner.

Amino Acid Motifs↗

PubMed1998

An alternatively spliced gene encoding a Y-box protein showing maternal expression and tissue-specific zygotic expression in the ascidian embryo.

An ascidian Y-box protein gene was cloned, designated as CiYB, which consists of a highly conserved cold shock domain and an auxiliary tail domain with alternating modules of acidic and basic amino acids. CiYB is a single copy gene in the ascidian genome. During oogenesis and early development, CiYB produces three different transcripts (CiYB1, CiYB2 and CiYB3) by alternate splicing. CiYB1 and CiYB2 were expressed during oogenesis, suggesting that they are recruited into maternal ribonucleoprotein particles. According to gel mobility shift assay, the CiYB1 protein has the ability to bind RNA. The sequence preference of RNA binding is similar to that of the Xenopus Y-box protein (FRGY2), which is a major component of the maternal messenger ribonucleoprotein particles (mRNP) in the oocyte. These results suggest that the ascidian Y-box protein may have an important role for masking and translational regulation of maternal mRNA. Furthermore, CiYB1, CiYB2 and CiYB3 were expressed zygotically in a tissue restricted manner. CiYB1 was expressed specifically in muscle precursor blastomeres and tail muscle cells suggesting its important role in muscle differentiation.

Age Factors↗

PubMedDate not supplied

The DAL10 gene from Norway spruce (Picea abies) belongs to a potentially gymnosperm-specific subclass of MADS-box genes and is specifically active in seed cones and pollen cones.

Transcription factors encoded by different members of the MADS-box gene family have evolved central roles in the regulation of reproductive organ development in the flowering plants, the angiosperms. Development of the stamens and carpels, the pollen- and seed-bearing organs, involves the B- and C-organ-identity MADS-box genes. B- and C-type gene orthologs with activities specifically in developing pollen- and seed-bearing organs are also present in the distantly related gymnosperms: the conifers and the gnetophytes. We now report on the characterization of DAL10, a novel MADS-box gene from the conifer Norway spruce, which unlike the B- and C-type conifer genes shows no distinct orthology relationship to any angiosperm gene or clade in phylogenetic analyses. Like the B- and C-type genes, it is active specifically in developing pollen cones and seed cones. In situ RNA localization experiments show DAL10 to be expressed in the cone axis, which carry the microsporophylls of the young pollen cone. In contrast, in the seed cone it is expressed both in the cone axis and in the bracts, which subtend the ovuliferous scales. Expression data and the phenotype of transgenic Arabidopsis plants expressing DAL10 suggest that the gene may act upstream to or in concert with the B- and C-type genes in the establishment of reproductive identity of developing cones.

Base Sequence↗

PubMed2003

Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA.

Modulation of the structure of a leader RNA to control formation of an intrinsic termination signal is a common mechanism for regulation of gene expression in bacteria. Expression of the S box genes in Gram-positive organisms is induced in response to limitation for methionine. We previously postulated that methionine availability is monitored by binding of a regulatory factor to the leader RNA and suggested that methionine or S-adenosylmethionine (SAM) could serve as the metabolic signal. In this study, we show that efficient termination of the S box leader region by bacterial RNA polymerase depends on SAM but not on methionine or other related compounds. We also show that SAM directly binds to and induces a conformational change in the leader RNA. Both binding of SAM and SAM-directed transcription termination were blocked by leader mutations that cause constitutive expression in vivo. Overproduction of SAM synthetase in Bacillus subtilis resulted in delay in induction of S box gene expression in response to methionine starvation, consistent with the hypothesis that SAM is the molecular effector in vivo. These results indicate that SAM concentration is sensed directly by the nascent transcript in the absence of a trans-acting factor.

5' Untranslated Regions↗

PubMed2002

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex.

The genomes of hyperthermophilic Archaea encode dozens of methylation guide, C/D box small RNAs that guide 2'-O-methylation of ribose to specific sites in rRNA and various tRNAs. The genes encoding the Sulfolobus homologues of eukaryotic proteins that are known to be present in C/D box small nucleolar ribonucleoprotein (snoRNP) complexes were cloned, and the proteins (aFIB, aNOP56, and aL7a) were expressed and purified. The purified proteins along with an in vitro transcript of the Sulfolobus sR1 small RNA were reconstituted in vitro, into an RNP complex. The order of assembly of the three proteins onto the RNA was aL7a, aNOP56, and aFIB. The complex was active in targeting S-adenosyl methionine (SAM)-dependent, site-specific 2'-O-methylation of ribose to a short fragment of ribosomal RNA (rRNA) that was complementary to the D box guide region of the sR1 small RNA. The presence of aFIB was essential for methylation; mutant proteins having amino acid replacements in the SAM-binding motif of aFIB were able to assemble into an RNP complex, but the resulting complexes were defective in methylation activity. These experiments define the minimal number of components and the conditions required to achieve in vitro RNA guide-directed 2'-O-methylation of ribose in a target RNA.

Amino Acid Motifs↗

PubMed2001

Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C' position of the C-capping box of alpha-helices.

The C' position of the C-capping box is the second residue outside of the helix. Statistical analysis of residue distribution at the C' position in the alpha-helices' C-capping box showed that different amino acid residues occur with different probabilities, with the strongest preference being for glycine. To understand the physico-chemical basis for this preference, we studied the effects that 17 amino acid substitutions at the C' position in an alpha-helix of ubiquitin have on the stability of this protein. We determined the following rank order of amino acid residues at the C' position with respect to their effect on the stability: Gly>His>Asn>Arg>Lys>Gln>Ala>Phe>Met>Ser>Asp>Glu>Trp>Thr>Pro>Ile>Val. The effect of the amino acid substitutions on the structure also was evaluated by comparing the (1)H-(15)N heteronuclear sequential quantum correlation spectra and showed no significant changes in the structures of the most stable (Gly) and the least stable (Val) variants. The obtained changes in stability highly correlate (r = 0.85) with the statistical distribution of the residues at the C' position indicating that the measured thermodynamic propensities are unbiased by secondary interactions. We also found that the measured thermodynamic propensities correlate well with the amide hydrogen exchange data on short model peptides (r = 0.85) and the calculated hydration of the peptide backbone (r = 0.88). These results combined with the changes in enthalpy and entropy of unfolding of ubiquitin variants suggest that dehydration of the peptide backbone plays a significant role in defining the thermodynamic propensity scale at the C' position of the C-capping box in alpha-helices. This propensity scale is useful for protein secondary structure predictions and protein design.

Amino Acid Substitution↗

PubMed2001

The SOCS box of suppressor of cytokine signaling-1 is important for inhibition of cytokine action in vivo.

Suppressor of Cytokine Signaling-1 (SOCS-1) is an essential physiological inhibitor of IFN-gamma signaling. Mice lacking this gene die in the early postnatal period from a disease characterized by hyperresponsiveness to endogenous IFN-gamma. The SOCS box is a C-terminal domain shared with over 30 other proteins that links SOCS proteins to an E3 ubiquitin ligase activity and the proteasome, but whether it contributes to inhibition of cytokine signaling is currently disputed. We have deleted only the SOCS box of the SOCS-1 gene in mice and show that such mice have an increased responsiveness to IFN-gamma and slowly develop a fatal inflammatory disease. These results demonstrate that deletion of the SOCS box leads to a partial loss of function of SOCS-1.

Animals↗

PubMed2003

Reversing established sepsis with antagonists of endogenous high-mobility group box 1.

Despite significant advances in intensive care therapy and antibiotics, severe sepsis accounts for 9% of all deaths in the United States annually. The pathological sequelae of sepsis are characterized by a systemic inflammatory response, but experimental therapeutics that target specific early inflammatory mediators [tumor necrosis factor (TNF) and IL-1beta] have not proven efficacious in the clinic. We recently identified high mobility group box 1 (HMGB1) as a late mediator of endotoxin-induced lethality that exhibits significantly delayed kinetics relative to TNF and IL-1beta. Here, we report that serum HMGB1 levels are increased significantly in a standardized model of murine sepsis, beginning 18 h after surgical induction of peritonitis. Specific inhibition of HMGB1 activity [with either anti-HMGB1 antibody (600 microg per mouse) or the DNA-binding A box (600 microg per mouse)] beginning as late as 24 h after surgical induction of peritonitis significantly increased survival (nonimmune IgG-treated controls = 28% vs. anti-HMGB1 antibody group = 72%, P < 0.03; GST control protein = 28% vs. A box = 68%, P < 0.03). Animals treated with either HMGB1 antagonist were protected against the development of organ injury, as evidenced by improved levels of serum creatinine and blood urea nitrogen. These observations demonstrate that specific inhibition of endogenous HMGB1 therapeutically reverses lethality of established sepsis indicating that HMGB1 inhibitors can be administered in a clinically relevant time frame.

Animals↗