scholarly journals Requirements for Septal Localization and Chromosome Segregation Activity of the DNA Translocase SftA from Bacillus subtilis

2017 ◽  
Vol 27 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Nina El Najjar ◽  
Christine Kaimer ◽  
Thomas Rösch ◽  
Peter L. Graumann
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Chromosome Segregation ◽  
Protein Interactions ◽  
Binding Motif ◽  
Cell Fractionation ◽  
Binding Studies ◽  
Mobility Shift ◽  
Septal Localization

<i>Bacillus subtilis</i> possesses 2 DNA translocases that affect late stages of chromosome segregation: SftA separates nonsegregated DNA prior to septum closure, while SpoIIIE rescues septum-entrapped DNA. We provide evidence that SftA is associated with the division machinery via a stretch of 47 amino acids within its N-terminus, suggesting that SftA is recruited by protein-protein interactions with a component of the division machinery. SftA was also recruited to mid-cell in the absence of its first 20 amino acids, which are proposed to contain a membrane-binding motif. Cell fractionation experiments showed that SftA can be found in the cytosolic fraction, and to a minor degree in the membrane fraction, showing that it is a soluble protein in vivo. The expression of truncated SftA constructs led to a dominant <i>sftA</i> deletion phenotype, even at very low induction rates of the truncated proteins, indicating that the incorporation of nonfunctional monomers into SftA hexamers abolishes functionality. Mobility shift experiments and surface plasmon binding studies showed that SftA binds to DNA in a cooperative manner, and demonstrated low ATPase activity when binding to short nucleotides rather than to long stretches of DNA.

scholarly journals Transcriptional regulation of the l-lactate permease gene lutP by the LutR repressor of Bacillus subtilis RO-NN-1

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2178-2189 ◽  
Author(s):  
Kuo-Chin Chiu ◽  
Chen-Jyun Lin ◽  
Gwo-Chyuan Shaw
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Binding Site ◽  
Repeat Sequence ◽  
Full Length ◽  
Binding Motif ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Reporter Gene Analysis

The Bacillus subtilis lutABC operon encodes three iron–sulfur-containing proteins required for l-lactate utilization and involved in biofilm formation. The transcriptional regulator LutR of the GntR family negatively controls lutABC expression. The lutP gene, which is situated immediately upstream of lutR, encodes an l-lactate permease. Here, we show that lutP expression can be strongly induced by l-lactate and is subject to partial catabolite repression by glucose. Disruption of the lutR gene led to a strong derepression of lutP and no further induction by l-lactate, suggesting that the LutR repressor can also negatively control lutP expression. Electrophoretic mobility shift assay revealed a LutR-binding site located downstream of the promoter of lutA or lutP and containing a consensus inverted repeat sequence 5′-TCATC-N1-GATGA-3′. Reporter gene analysis showed that deletion of each LutR-binding site caused a strong derepression of lutA or lutP. These results indicated that these two LutR-binding sites can function as operators in vivo. Moreover, deletion analysis identified a DNA segment upstream of the lutP promoter to be important for lutP expression. In contrast to the truncated LutR of laboratory strains 168 and PY79, the full-length LutR of the undomesticated strain RO-NN-1, and probably many other B. subtilis strains, can directly and negatively regulate lutP transcription. The absence or presence of the N-terminal 21 aa of the full-length LutR, which encompass a small part of the predicted winged helix–turn–helix DNA-binding motif, may probably alter the DNA-binding specificity or affinity of LutR.

scholarly journals Genetic and Biochemical Analysis of the Interaction of Bacillus subtilis CodY with Branched-Chain Amino Acids

2009 ◽  
Vol 191 (22) ◽  
pp. 6865-6876 ◽  
Author(s):  
Anuradha C. Villapakkam ◽  
Luke D. Handke ◽  
Boris R. Belitsky ◽  
Vladimir M. Levdikov ◽  
Anthony J. Wilkinson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Branched Chain Amino Acids ◽  
Site Directed Mutagenesis ◽  
Binding Studies ◽  
Hydrophobic Pocket ◽  
Isoleucine Leucine ◽  
Branched Chain

ABSTRACT Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals Mutational Analysis of the Cy Motif from p21 Reveals Sequence Degeneracy and Specificity for Different Cyclin-Dependent Kinases

2001 ◽  
Vol 21 (15) ◽  
pp. 4868-4874 ◽  
Author(s):  
James A. Wohlschlegel ◽  
Brian T. Dwyer ◽  
David Y. Takeda ◽  
Anindya Dutta
Keyword(s):  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Cyclin E ◽  
Cyclin A ◽  
Binding Motif ◽  
Binding Studies ◽  
Alanine Scanning Mutagenesis ◽  
Cyclin Dependent Kinases

ABSTRACT Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.

Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

2021 ◽  
Author(s):  
Babu Sudhamalla ◽  
Anirban Roy ◽  
Soumen Barman ◽  
Jyotirmayee Padhan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Unnatural Amino Acids ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

scholarly journals Slitrk2 controls excitatory synapse development via PDZ-mediated protein interactions

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kyung Ah Han ◽  
Jinhu Kim ◽  
Hyeonho Kim ◽  
Dongwook Kim ◽  
Dongseok Lim ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Intracellular Signaling ◽  
Hippocampal Neurons ◽  
Pdz Domain ◽  
Protein Tyrosine Phosphatases ◽  
Excitatory Synapse ◽  
Receptor Protein ◽  
Binding Motif ◽  
Synapse Development

AbstractMembers of the Slitrk (Slit- and Trk-like protein) family of synaptic cell-adhesion molecules control excitatory and inhibitory synapse development through isoform-dependent extracellular interactions with leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs). However, how Slitrks participate in activation of intracellular signaling pathways in postsynaptic neurons remains largely unknown. Here we report that, among the six members of the Slitrk family, only Slitrk2 directly interacts with the PDZ domain-containing excitatory scaffolds, PSD-95 and Shank3. The interaction of Slitrk2 with PDZ proteins is mediated by the cytoplasmic COOH-terminal PDZ domain-binding motif (Ile-Ser-Glu-Leu), which is not found in other Slitrks. Mapping analyses further revealed that a single PDZ domain of Shank3 is responsible for binding to Slitrk2. Slitrk2 forms in vivo complexes with membrane-associated guanylate kinase (MAGUK) family proteins in addition to PSD-95 and Shank3. Intriguingly, in addition to its role in synaptic targeting in cultured hippocampal neurons, the PDZ domain-binding motif of Slitrk2 is required for Slitrk2 promotion of excitatory synapse formation, transmission, and spine development in the CA1 hippocampal region. Collectively, our data suggest a new molecular mechanism for conferring isoform-specific regulatory actions of the Slitrk family in orchestrating intracellular signal transduction pathways in postsynaptic neurons.

scholarly journals Loading of DNA-Binding Factors to an Erythroid Enhancer

2000 ◽  
Vol 20 (6) ◽  
pp. 1993-2003 ◽  
Author(s):  
Shau-Ching Wen ◽  
Karim Roder ◽  
Kuang-Yu Hu ◽  
Irene Rombel ◽  
Narender R. Gavva ◽  
...  
Keyword(s):  
Regulatory Element ◽  
K562 Cells ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Binding Studies ◽  
Mobility Shift ◽  
Specific Expression ◽  
Factor Binding ◽  
Globin Promoter

ABSTRACT The HS-40 enhancer is the major cis-acting regulatory element responsible for the developmental stage- and erythroid lineage-specific expression of the human α-like globin genes, the embryonic ζ and the adult α2/α/1. A model has been proposed in which competitive factor binding at one of the HS-40 motifs, 3′-NA, modulates the capability of HS-40 to activate the embryonic ζ-globin promoter. Furthermore, this modulation was thought to be mediated through configurational changes of the HS-40 enhanceosome during development. In this study, we have further investigated the molecular basis of this model. First, human erythroid K562 cells stably integrated with various HS-40 mutants cis linked to a human α-globin promoter-growth hormone hybrid gene were analyzed by genomic footprinting and expression analysis. By the assay, we demonstrate that factors bound at different motifs of HS-40 indeed act in concert to build a fully functional enhanceosome. Thus, modification of factor binding at a single motif could drastically change the configuration and function of the HS-40 enhanceosome. Second, a specific 1-bp, GC→TA mutation in the 3′-NA motif of HS-40, 3′-NA(II), has been shown previously to cause significant derepression of the embryonic ζ-globin promoter activity in erythroid cells. This derepression was hypothesized to be regulated through competitive binding of different nuclear factors, in particular AP1 and NF-E2, to the 3′-NA motif. By gel mobility shift and transient cotransfection assays, we now show that 3′-NA(II) mutation completely abolishes the binding of small MafK homodimer. Surprisingly, NF-E2 as well as AP1 can still bind to the 3′-NA(II) sequence. The association constants of both NF-E2 and AP1 are similar to their interactions with the wild-type 3′-NA motif. However, the 3′-NA(II) mutation causes an approximately twofold reduction of the binding affinity of NF-E2 factor to the 3′-NA motif. This reduction of affinity could be accounted for by a twofold-higher rate of dissociation of the NF-E2–3′-NA(II) complex. Finally, we show by chromatin immunoprecipitation experiments that only binding of NF-E2, not AP1, could be detected in vivo in K562 cells around the HS-40 region. These data exclude a role for AP1 in the developmental regulation of the human α-globin locus via the 3′-NA motif of HS-40 in embryonic/fetal erythroid cells. Furthermore, extrapolation of the in vitro binding studies suggests that factors other than NF-E2, such as the small Maf homodimers, are likely involved in the regulation of the HS-40 function in vivo.

scholarly journals Molecular Mechanisms Underlying the Positive Stringent Response of the Bacillus subtilis ilv-leu Operon, Involved in the Biosynthesis of Branched-Chain Amino Acids

2008 ◽  
Vol 190 (18) ◽  
pp. 6134-6147 ◽  
Author(s):  
Shigeo Tojo ◽  
Takenori Satomura ◽  
Kanako Kumamoto ◽  
Kazutake Hirooka ◽  
Yasutaro Fujita
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Stringent Response ◽  
Branched Chain Amino Acids ◽  
Base Substitution ◽  
Branched Chain

ABSTRACT Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5′ end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

scholarly journals Enzymic determination of branched-chain amino acids and 2-oxoacids in rat tissues. Transfer of 2-oxoacids from skeletal muscle to liver in vivo

1980 ◽  
Vol 188 (3) ◽  
pp. 705-713 ◽  
Author(s):  
G Livesey ◽  
P Lund
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Bacillus Subtilis ◽  
Branched Chain Amino Acids ◽  
Rat Tissues ◽  
Arterial Blood ◽  
Branched Chain ◽  
Arteriovenous Difference

1. A procedure is described for the purification of leucine dehydrogenase (EC 1.4.1.9) from Bacillus subtilis. 2. The preparation is suitable for the quantitative assay of branched-chain amino acids and their 2-oxoacid analogues. 3. The content of total branched-chain 2-oxoacids in freeze-clamped liver, kidney, heart or mammary gland of fed rats is less than 5 nmol/g fresh wt. Higher amounts are present in skeletal muscle and arterial blood (25 +/- 4 nmol per g fresh wt., and 33 +/- 6 nmol per ml respectively; means +/- S.D. of 3 and 11 animals respectively). The values are not significantly affected by starvation for 24 h. 4. Arteriovenous difference measurements show that considerable amounts of branched-chain 2-oxoacids are released by skeletal muscle into the circulation and similar amounts are removed by the liver (about 1 mmol/24 h in a 400 g rat).

scholarly journals In Vivo Effect of Mutations at the PRPP Binding Site of the Bacillus subtilis Purine Repressor

2003 ◽  
Vol 185 (22) ◽  
pp. 6728-6731 ◽  
Author(s):  
Pekka Rappu ◽  
Terhi Pullinen ◽  
Pekka Mäntsälä
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Binding Motif

ABSTRACT The Bacillus subtilis PurR mediates adenine repression and guanosine induction of purA. PRPP inhibits binding of PurR to DNA in vitro. Mutations in the PRPP binding motif of PurR caused strong repression regardless of purine exclusions or additions, establishing the role of PRPP as regulator of PurR.

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