scholarly journals Mapping of the interactions between partition proteins Delta and Omega of plasmid pSM19035 from Streptococcus pyogenes

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1009-1020 ◽  
Author(s):  
Michal Dmowski ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Streptococcus Pyogenes ◽  
Dna Sequences ◽  
Copy Number ◽  
Terminal Part ◽  
Partition System ◽  
Plasmid Partition ◽  
Nucleoprotein Complex ◽  
Interaction Domains

Formation of the segrosome, a nucleoprotein complex crucial for proper functioning of plasmid partition systems, involves interactions between specific partition proteins (ParA-like and ParB-like), ATP and specific DNA sequences (the centromeric sites). Although partition systems have been studied for many years, details of the segrosome formation are not yet clear. Organization of the pSM19035-encoded partition system is unique; in contrast with other known par systems, here, the δ and ω genes do not constitute an operon. Moreover, Omega [a ParB-like protein which has a Ribbon-Helix-Helix (RHH) structure] recognizes multiple centromeric sequences located in the promoters of δ, ω and copS (copy-number control gene). The ParA-like protein Delta is a Walker-type ATPase. In this work, we identify the interaction domains and requirements for dimerization and hetero-interactions of the Delta and Omega proteins of pSM19035 plasmid. The RHH structures are involved in Omega dimerization in vivo and its N-terminal unstructured part is indispensable for association with Delta, both in vivo and in vitro. Omega does not need to form dimers to interact with Delta. ATP binding is not required for Delta dimerization but is important for interaction with Omega in vivo. The in vitro interaction between Delta and Omega depends on ATP but does not require the presence of specific DNA segments (the centromere) recognized by Omega. The C-terminal part of the Delta protein (aa 198–284) is indispensable for interaction with Omega. Delta most probably interacts with Omega as a dimer since two amino acid substitutions in a conserved region between the A′ and B motifs abolish both the dimerization of Delta and its interaction with Omega.

scholarly journals Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

2015 ◽  
Vol 112 (51) ◽  
pp. E7055-E7064 ◽  
Author(s):  
Longhua Hu ◽  
Anthony G. Vecchiarelli ◽  
Kiyoshi Mizuuchi ◽  
Keir C. Neuman ◽  
Jian Liu
Keyword(s):  
Copy Number ◽  
Detailed Mechanism ◽  
Ratchet Mechanism ◽  
Partition System ◽  
Plasmid Segregation ◽  
Emergent Phenomenon ◽  
Mechanochemical Coupling ◽  
Low Copy Number

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA–nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos—an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

scholarly journals The Transcriptional Regulator CpsY Is Important for Innate Immune Evasion in Streptococcus pyogenes

2016 ◽  
Vol 85 (3) ◽  
Author(s):  
Luis A. Vega ◽  
Kayla M. Valdes ◽  
Ganesh S. Sundar ◽  
Ashton T. Belew ◽  
Emrul Islam ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Immune Evasion ◽  
Immune Cell ◽  
Group A Streptococcus ◽  
Transcriptional Regulator ◽  
Innate Immune ◽  
Content Type ◽  
Human Host

ABSTRACTAs an exclusively human pathogen,Streptococcus pyogenes(the group A streptococcus [GAS]) has specifically adapted to evade host innate immunity and survive in multiple tissue niches, including blood. GAS can overcome the metabolic constraints of the blood environment and expresses various immunomodulatory factors necessary for survival and immune cell resistance. Here we present our investigation of one such factor, the predicted LysR family transcriptional regulator CpsY. The encoding gene,cpsY, was initially identified as being required for GAS survival in a transposon-site hybridization (TraSH) screen in whole human blood. CpsY is homologous with transcriptional regulators ofStreptococcus mutans(MetR),Streptococcus iniae(CpsY), andStreptococcus agalactiae(MtaR) that regulate methionine transport, amino acid metabolism, resistance to neutrophil-mediated killing, and survivalin vivo. Our investigation indicated that CpsY is involved in GAS resistance to innate immune cells of its human host. However, GAS CpsY does not manifest thein vitrophenotypes of its homologs in other streptococcal species. GAS CpsY appears to regulate a small set of genes that is markedly different from the regulons of its homologs. The differential expression of these genes depends on the growth medium, and CpsY modestly influences their expression. The GAS CpsY regulon includes known virulence factors (mntE,speB,spd,nga[spn],prtS[SpyCEP], andsse) and cell surface-associated factors of GAS (emm1,mur1.2,sibA[cdhA], andM5005_Spy0500). Intriguingly, the loss of CpsY in GAS does not result in virulence defects in murine models of infection, suggesting that CpsY function in immune evasion is specific to the human host.

scholarly journals Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2276-2285 ◽  
Author(s):  
Maria De La Luz Sierra ◽  
Paola Gasperini ◽  
Peter J. McCormick ◽  
Jinfang Zhu ◽  
Giovanna Tosato
Keyword(s):  
Bone Marrow ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Cell Function ◽  
Myeloid Cell ◽  
Cxcr4 Expression ◽  
Negative Regulator ◽  
Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

scholarly journals ParB of Pseudomonas aeruginosa: Interactions with Its Partner ParA and Its Target parS and Specific Effects on Bacterial Growth

2004 ◽  
Vol 186 (20) ◽  
pp. 6983-6998 ◽  
Author(s):  
Aneta A. Bartosik ◽  
Krzysztof Lasocki ◽  
Jolanta Mierzejewska ◽  
Christopher M. Thomas ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Streptomyces Coelicolor ◽  
Consensus Sequence ◽  
Terminal Part ◽  
Dimerization Domain ◽  
Interaction Domain ◽  
C Terminus ◽  
Cell Components

ABSTRACT The par genes of Pseudomonas aeruginosa have been studied to increase the understanding of their mechanism of action and role in the bacterial cell. Key properties of the ParB protein have been identified and are associated with different parts of the protein. The ParB- ParB interaction domain was mapped in vivo and in vitro to the C-terminal 56 amino acids (aa); 7 aa at the C terminus play an important role. The dimerization domain of P. aeruginosa ParB is interchangeable with the dimerization domain of KorB from plasmid RK2 (IncP1 group). The C-terminal part of ParB is also involved in ParB-ParA interactions. Purified ParB binds specifically to DNA containing a putative parS sequence based on the consensus sequence found in the chromosomes of Bacillus subtilis, Pseudomonas putida, and Streptomyces coelicolor. The overproduction of ParB was shown to inhibit the function of genes placed near parS. This “silencing” was dependent on the parS sequence and its orientation. The overproduction of P. aeruginosa ParB or its N-terminal part also causes inhibition of the growth of P. aeruginosa and P. putida but not Escherichia coli cells. Since this inhibitory determinant is located well away from ParB segments required for dimerization or interaction with the ParA counterpart, this result may suggest a role for the N terminus of P. aeruginosa ParB in interactions with host cell components.

scholarly journals Triple-Helical DNA in Drosophila Heterochromatin

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 227 ◽  
Author(s):  
Eduardo Gorab
Keyword(s):  
Nucleic Acids ◽  
Dna Sequences ◽  
Detection Methods ◽  
Triplex Dna ◽  
Thiazole Orange ◽  
Chromosomal Dna ◽  
Mitotic Chromosomes ◽  
Satellite Repeats

Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro, their occurrence in vivo has been strengthened by increasing evidence, although most remain circumstantial particularly for triplex DNA. Here, different approaches were employed to specify triple-stranded DNA sequences in the Drosophila melanogaster chromosomes. Antibodies to triplex nucleic acids, previously characterized, bind to centromeric regions of mitotic chromosomes and also to the polytene section 59E of mutant strains carrying the brown dominant allele, indicating that AAGAG tandem satellite repeats are triplex-forming sequences. The satellite probe hybridized to AAGAG-containing regions omitting chromosomal DNA denaturation, as expected, for the intra-molecular triplex DNA formation model in which single-stranded DNA coexists with triplexes. In addition, Thiazole Orange, previously described as capable of reproducing results obtained by antibodies to triple-helical DNA, binds to AAGAG repeats in situ thus validating both detection methods. Unusual phenotype and nuclear structure exhibited by Drosophila correlate with the non-canonical conformation of tandem satellite arrays. From the approaches that lead to the identification of triple-helical DNA in chromosomes, facilities particularly provided by Thiazole Orange use may broaden the investigation on the occurrence of triplex DNA in eukaryotic genomes.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals LiquidCNA: tracking subclonal evolution from longitudinal liquid biopsies using somatic copy number alterations

2021 ◽  
Author(s):  
Eszter Lakatos ◽  
Helen Hockings ◽  
Maximilian Mossner ◽  
Michelle Lockley ◽  
Trevor A. Graham
Keyword(s):  
Copy Number ◽  
Evolutionary Dynamics ◽  
Cost Effective ◽  
Copy Number Alterations ◽  
Liquid Biopsies ◽  
Metastatic Lung ◽  
Low Pass ◽  
Somatic Copy Number Alterations

AbstractCell-free DNA (cfDNA) measured via liquid biopsies provides a way for minimally-invasive monitoring of tumour evolutionary dynamics during therapy. Here we present liquidCNA, a method to track subclonal evolution from longitudinally collected cfDNA samples based on somatic copy number alterations (SCNAs). LiquidCNA utilises SCNA profiles derived through cost-effective low-pass whole genome sequencing to automatically and simultaneously genotype and quantify the size of the dominant subclone without requiring prior knowledge of the genetic identity of the emerging clone. We demonstrate the accuracy of liquidCNA in synthetically generated sample sets and in vitro and in silico mixtures of cancer cell lines. Application in vivo in patients with metastatic lung cancer reveals the progressive emergence of a novel tumour sub-population. LiquidCNA is straightforward to use, computationally inexpensive and enables continuous monitoring of subclonal evolution to understand and control therapy-induced resistance.

Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas

1989 ◽  
Vol 9 (6) ◽  
pp. 2464-2476
Author(s):  
M Cockell ◽  
B J Stevenson ◽  
M Strubin ◽  
O Hagenbüchle ◽  
P K Wellauer
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Binding Activity ◽  
Alpha Amylase ◽  
Dna Binding Activity ◽  
A Cell ◽  
Flanking Regions

Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription

1984 ◽  
Vol 4 (1) ◽  
pp. 133-141
Author(s):  
J Brady ◽  
M Radonovich ◽  
M Thoren ◽  
G Das ◽  
N P Salzman
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Simian Virus ◽  
Upstream Promoter ◽  
Upstream Promoter Sequence

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294.

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