scholarly journals Induced DNA bending by unique dimerization of HigA antitoxin

IUCrJ ◽  
2020 ◽  
Vol 7 (4) ◽  
pp. 748-760
Author(s):  
Jin-Young Park ◽  
Hyo Jung Kim ◽  
Chinar Pathak ◽  
Hye-Jin Yoon ◽  
Do-Hee Kim ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Gene Organization ◽  
Bacterial Toxin ◽  
Type Ii ◽  
Dimerization Domain ◽  
Tb Treatment ◽  
C Terminus ◽  
New Antibiotics ◽  
Toxin Protein ◽  
Promoter Dna

The bacterial toxin–antitoxin (TA) system regulates cell growth under various environmental stresses. Mycobacterium tuberculosis, the causative pathogen of tuberculosis (TB), has three HigBA type II TA systems with reverse gene organization, consisting of the toxin protein HigB and labile antitoxin protein HigA. Most type II TA modules are transcriptionally autoregulated by the antitoxin itself. In this report, we first present the crystal structure of the M. tuberculosis HigA3 antitoxin (MtHigA3) and MtHigA3 bound to its operator DNA complex. We also investigated the interaction between MtHigA3 and DNA using NMR spectroscopy. The MtHigA3 antitoxin structure is a homodimer that contains a structurally well conserved DNA-binding domain at the N-terminus and a dimerization domain at the C-terminus. Upon comparing the HigA homologue structures, a distinct difference was found in the C-terminal region that possesses the β-lid, and diverse orientations of two helix–turn–helix (HTH) motifs from HigA homologue dimers were observed. The structure of MtHigA3 bound to DNA reveals that the promoter DNA is bound to two HTH motifs of the MtHigA3 dimer presenting 46.5° bending, and the distance between the two HTH motifs of each MtHigA3 monomer was increased in MtHigA3 bound to DNA. The β-lid, which is found only in the tertiary structure of MtHigA3 among the HigA homologues, causes the formation of a tight dimerization network and leads to a unique arrangement for dimer formation that is related to the curvature of the bound DNA. This work could contribute to the understanding of the HigBA system of M. tuberculosis at the atomic level and may contribute to the development of new antibiotics for TB treatment.

scholarly journals Defining the mRNA recognition signature of a bacterial toxin protein

2015 ◽  
Vol 112 (45) ◽  
pp. 13862-13867 ◽  
Author(s):  
Marc A. Schureck ◽  
Jack A. Dunkle ◽  
Tatsuya Maehigashi ◽  
Stacey J. Miles ◽  
Christine M. Dunham
Keyword(s):  
Important Determinant ◽  
Stringent Response ◽  
Bacterial Toxin ◽  
Type Ii ◽  
X Ray ◽  
The Third ◽  
Inhibition Of Growth ◽  
Toxin Protein ◽  
A Site ◽  
Multiple Type

Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosome-dependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. Here, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop to recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide.

scholarly journals Interactions of the Streptococcus pneumoniae Toxin-Antitoxin RelBE Proteins with Their Target DNA

2021 ◽  
Vol 9 (4) ◽  
pp. 851
Author(s):  
Inmaculada Moreno-Córdoba ◽  
Wai-Ting Chan ◽  
Concha Nieto ◽  
Manuel Espinosa
Keyword(s):  
Streptococcus Pneumoniae ◽  
Self Regulation ◽  
Mrna Translation ◽  
Dna Supercoiling ◽  
Bacterial Toxin ◽  
Type Ii ◽  
Cellular Functions ◽  
Dna Backbone ◽  
Different Strains ◽  
Palindromic Sequences

Type II bacterial toxin-antitoxin (TA) systems are found in most bacteria, archaea, and mobile genetic elements. TAs are usually found as a bi-cistronic operon composed of an unstable antitoxin and a stable toxin that targets crucial cellular functions like DNA supercoiling, cell-wall synthesis or mRNA translation. The type II RelBE system encoded by the pathogen Streptococcus pneumoniae is highly conserved among different strains and participates in biofilm formation and response to oxidative stress. Here, we have analyzed the participation of the RelB antitoxin and the RelB:RelE protein complex in the self-regulation of the pneumococcal relBE operon. RelB acted as a weak repressor, whereas RelE performed the role of a co-repressor. By DNA footprinting experiments, we show that the proteins bind to a region that encompasses two palindromic sequences that are located around the −10 sequences of the single promoter that directs the synthesis of the relBE mRNA. High-resolution footprinting assays showed the distribution of bases whose deoxyriboses are protected by the bound proteins, demonstrating that RelB and RelB:RelE contacted the DNA backbone on one face of the DNA helix and that these interactions extended beyond the palindromic sequences. Our findings suggest that the binding of the RelBE proteins to its DNA target would lead to direct inhibition of the binding of the host RNA polymerase to the relBE promoter.

scholarly journals Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters

2007 ◽  
Vol 73 (18) ◽  
pp. 5711-5715 ◽  
Author(s):  
Sung Kuk Lee ◽  
Howard H. Chou ◽  
Brian F. Pfleger ◽  
Jack D. Newman ◽  
Yasuo Yoshikuni ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Cross Talk ◽  
Biological Systems ◽  
Wild Type ◽  
Dimerization Domain ◽  
Expression Levels ◽  
Inducible Promoters ◽  
C Terminus ◽  
Increased Sensitivity ◽  
Better Than

ABSTRACT Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (PBAD) system that is more compatible with IPTG (isopropyl-β-d-1-thiogalactopyranoside) induction of a lactose-inducible (Plac) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.

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.

Differences in gene organization between type I and type II crustins in the morotoge shrimp, Pandalopsis japonica

2013 ◽  
Vol 35 (4) ◽  
pp. 1176-1184 ◽  
Author(s):  
Bokwang Kim ◽  
Meesun Kim ◽  
Ah Ran Kim ◽  
Myunggi Yi ◽  
Jung-Hwa Choi ◽  
...  
Keyword(s):  
Gene Organization ◽  
Type I ◽  
Type Ii

scholarly journals Genetically modified human type II collagen for N- and C-terminal covalent tagging

2018 ◽  
Vol 96 (2) ◽  
pp. 204-211
Author(s):  
Andrew Wieczorek ◽  
Clara K. Chan ◽  
Suzana Kovacic ◽  
Cindy Li ◽  
Thomas Dierks ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Connective Tissue Diseases ◽  
Sulfhydryl Group ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Structural Protein ◽  
Connective Tissues ◽  
Human Type ◽  
C Terminus ◽  
Transmission Electron

Collagen is the predominant structural protein in vertebrates, where it contributes to connective tissues and the ECM; it is also widely used in biomaterials and tissue engineering. Dysfunction of this protein and its processing can lead to a wide variety of developmental disorders and connective tissue diseases. Recombinantly engineering the protein is challenging due to post-translational modifications generally required for its stability and secretion from cells. Introducing end labels into the protein is problematic, because the N- and C-termini of the physiologically relevant tropocollagen lie internal to the initially flanking N- and C-propeptide sequences. Here, we introduce mutations into human type II procollagen in a manner that addresses these concerns and purify the recombinant protein from a stably transfected HT1080 human fibrosarcoma cell line. Our approach introduces chemically addressable groups into the N- and C-telopeptide termini of tropocollagen. Simultaneous overexpression of formylglycine generating enzyme (FGE) allows the endogenous production of an aldehyde tag in a defined, substituted sequence in the N terminus of the mutated collagen, whereas the C-terminus of each chain presents a sulfhydryl group from an introduced cysteine. These modifications are designed to enable specific covalent end-labelling of collagen. We find that the doubly mutated protein folds and is secreted from cells. Higher order assembly into well-ordered collagen fibrils is demonstrated through transmission electron microscopy. Chemical tagging of thiols is successful; however, background from endogenous aldehydes present in wild-type collagen has thus far obscured the desired specific N-terminal labelling. Strategies to overcome this challenge are proposed.

scholarly journals Toxin-Antitoxin Genes of the Gram-Positive Pathogen Streptococcus pneumoniae: So Few and Yet So Many

2012 ◽  
Vol 76 (4) ◽  
pp. 773-791 ◽  
Author(s):  
Wai Ting Chan ◽  
Inma Moreno-Córdoba ◽  
Chew Chieng Yeo ◽  
Manuel Espinosa
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Bacterial Infections ◽  
Host Cells ◽  
Molecular Characteristics ◽  
Type Ii ◽  
Pneumococcal Infections ◽  
Interesting Phenomenon ◽  
Content Type ◽  
Toxin Protein

SUMMARYPneumococcal infections cause up to 2 million deaths annually and raise a large economic burden and thus constitute an important threat to mankind. Because of the increase in the antibiotic resistance ofStreptococcus pneumoniaeclinical isolates, there is an urgent need to find new antimicrobial approaches to triumph over pneumococcal infections. Toxin-antitoxin (TA) systems (TAS), which are present in most living bacteria but not in eukaryotes, have been proposed as an effective strategy to combat bacterial infections. Type II TAS comprise a stable toxin and a labile antitoxin that form an innocuous TA complex under normal conditions. Under stress conditions, TA synthesis will be triggered, resulting in the degradation of the labile antitoxin and the release of the toxin protein, which would poison the host cells. The three functional chromosomal TAS fromS. pneumoniaethat have been studied as well as their molecular characteristics are discussed in detail in this review. Furthermore, a meticulous bioinformatics search has been performed for 48 pneumococcal genomes that are found in public databases, and more putative TAS, homologous to well-characterized ones, have been revealed. Strikingly, several unusual putative TAS, in terms of components and genetic organizations previously not envisaged, have been discovered and are further discussed. Previously, we reported a novel finding in which a unique pneumococcal DNA signature, the BOX element, affected the regulation of the pneumococcalyefM-yoeBTAS. This BOX element has also been found in some of the other pneumococcal TAS. In this review, we also discuss possible relationships between some of the pneumococcal TAS with pathogenicity, competence, biofilm formation, persistence, and an interesting phenomenon called bistability.

scholarly journals LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor

2019 ◽  
Vol 47 (12) ◽  
pp. 6369-6385
Author(s):  
Jia-Yi Fan ◽  
Qian Huang ◽  
Quan-Quan Ji ◽  
En-Duo Wang
Keyword(s):  
Tertiary Structure ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Type I ◽  
Type Ii ◽  
Specific Domain ◽  
Recognition Mechanism ◽  
Variable Loop

Abstract Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

scholarly journals Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (9) ◽  
pp. 2681-2697 ◽  
Author(s):  
Kenneth Moss ◽  
Andrew Helm ◽  
Yun Lu ◽  
Alvina Bragin ◽  
William R. Skach
Keyword(s):  
Endoplasmic Reticulum ◽  
Structural Features ◽  
Endoplasmic Reticulum Membrane ◽  
Type I ◽  
Type Ii ◽  
Hydrophobic Residues ◽  
P Glycoprotein ◽  
C Terminus ◽  
Signal Anchor ◽  
Hydrophilic Residues

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single- and a double-spanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8.

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