scholarly journals Structural mechanism of transcription regulation of the Staphylococcus aureus multidrug efflux operon mepRA by the MarR family repressor MepR

2013 ◽  
Vol 42 (4) ◽  
pp. 2774-2788 ◽  
Author(s):  
Ivan Birukou ◽  
Susan M. Seo ◽  
Bryan D. Schindler ◽  
Glenn W. Kaatz ◽  
Richard G. Brennan
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Conformational Changes ◽  
Efflux Pump ◽  
Binding Activity ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Structural Mechanism ◽  
Dna Binding Activity ◽  
Complex Structural

Abstract The multidrug efflux pump MepA is a major contributor to multidrug resistance in Staphylococcus aureus. MepR, a member of the multiple antibiotic resistance regulator (MarR) family, represses mepA and its own gene. Here, we report the structure of a MepR–mepR operator complex. Structural comparison of DNA-bound MepR with ‘induced’ apoMepR reveals the large conformational changes needed to allow the DNA-binding winged helix-turn-helix motifs to interact with the consecutive major and minor grooves of the GTTAG signature sequence. Intriguingly, MepR makes no hydrogen bonds to major groove nucleobases. Rather, recognition-helix residues Thr60, Gly61, Pro62 and Thr63 make sequence-specifying van der Waals contacts with the TTAG bases. Removing these contacts dramatically affects MepR–DNA binding activity. The wings insert into the flanking minor grooves, whereby residue Arg87, buttressed by Asp85, interacts with the O2 of T4 and O4′ ribosyl oxygens of A23 and T4. Mutating Asp85 and Arg87, both conserved throughout the MarR family, markedly affects MepR repressor activity. The His14′:Arg59 and Arg10′:His35:Phe108 interaction networks stabilize the DNA-binding conformation of MepR thereby contributing significantly to its high affinity binding. A structure-guided model of the MepR–mepA operator complex suggests that MepR dimers do not interact directly and cooperative binding is likely achieved by DNA-mediated allosteric effects.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

scholarly journals The glucosaminidase domain of Atl – the major Staphylococcus aureus autolysin – has DNA ‐binding activity

2014 ◽  
Vol 3 (2) ◽  
pp. 247-256 ◽  
Author(s):  
Inês R. Grilo ◽  
Ana Madalena Ludovice ◽  
Alexander Tomasz ◽  
Hermínia Lencastre ◽  
Rita G. Sobral
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Binding Activity ◽  
Dna Binding Activity

scholarly journals Response of Staphylococcus aureus to Salicylate Challenge

2006 ◽  
Vol 189 (1) ◽  
pp. 220-227 ◽  
Author(s):  
James T. Riordan ◽  
Arunachalam Muthaiyan ◽  
Wayne Van Voorhies ◽  
Christopher T. Price ◽  
James E. Graham ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Resistance ◽  
Transcriptome Analysis ◽  
Target Genes ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Antibiotic Target ◽  
Unique Mechanism ◽  
Formate Metabolism

ABSTRACT Growth of Staphylococcus aureus with the nonsteroidal anti-inflammatory salicylate reduces susceptibility of the organism to multiple antimicrobials. Transcriptome analysis revealed that growth of S. aureus with salicylate leads to the induction of genes involved with gluconate and formate metabolism and represses genes required for gluconeogenesis and glycolysis. In addition, salicylate induction upregulates two antibiotic target genes and downregulates a multidrug efflux pump gene repressor (mgrA) and sarR, which represses a gene (sarA) important for intrinsic antimicrobial resistance. We hypothesize that these salicylate-induced alterations jointly represent a unique mechanism that allows S. aureus to resist antimicrobial stress and toxicity.

Conformational changes in bacteriophage Ø 29 connector prevents DNA-binding activity

1990 ◽  
Vol 213 (2) ◽  
pp. 263-273 ◽  
Author(s):  
Lucía Herranz ◽  
Joan Bordas ◽  
Elisabeth Towns-Andrews ◽  
Enrique Mendez ◽  
Pilar Usobiaga ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Changes ◽  
Binding Activity ◽  
O 29 ◽  
Dna Binding Activity

scholarly journals MepR, a Repressor of the Staphylococcus aureus MATE Family Multidrug Efflux Pump MepA, Is a Substrate-Responsive Regulatory Protein

2006 ◽  
Vol 50 (4) ◽  
pp. 1276-1281 ◽  
Author(s):  
Glenn W. Kaatz ◽  
Carmen E. DeMarco ◽  
Susan M. Seo
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Multidrug Efflux ◽  
Recognition Sequence ◽  
Multidrug Efflux Pump ◽  
Mobility Shift ◽  
Repressive Effect ◽  
Operator Interaction

ABSTRACT The mepRAB gene cluster of Staphylococcus aureus encodes a MarR family repressor (MepR; known to repress mepA expression), a MATE family multidrug efflux pump (MepA), and a protein of unknown function (MepB). In this report, we show that MepR also is autoregulatory, repressing the expression of its own gene. Exposure of strains containing a mepR::lacZ fusion with mepR provided in trans under the control of an inducible promoter, or a mepA::lacZ fusion alone, to subinhibitory concentrations of MepA substrates resulted in variably increased expression mainly of mepA. Mobility shift assays revealed that MepR binds upstream of mepR and mepA, with an apparently higher affinity for the mepA binding site. MepA substrates abrogated MepR binding to each site in a differential manner, with the greatest effect observed on the MepR-mepA operator interaction. DNase I footprinting identified precise binding sites which included promoter motifs, inverted repeats, and transcription start sites for mepR and mepA, as well as a conserved GTTAG motif, which may be a signature recognition sequence for MepR. Analogous to other multidrug efflux pump regulatory proteins such as QacR, the substrate-MepR interaction likely results in its dissociation from its mepA, and in a more limited fashion its mepR, operator sites and relief of its repressive effect. The enhanced effect of substrates on mepA compared to mepR expression, and on the MepR-mepA operator interaction, results in significant relief of mepA and relative maintenance of mepR repression, leading to increased MepA protein unimpeded by MepR when the need for detoxification exists.

scholarly journals Fluoroquinolone Efflux by the Plasmid-Mediated Multidrug Efflux Pump QacB Variant QacBIII in Staphylococcus aureus

2010 ◽  
Vol 54 (10) ◽  
pp. 4107-4111 ◽  
Author(s):  
Hidemasa Nakaminami ◽  
Norihisa Noguchi ◽  
Masanori Sasatsu
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Efflux Pump ◽  
Amino Acid Sequences ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Substrate Specificities

ABSTRACT Plasmids that carry the multidrug efflux genes qacA and qacB are widely distributed in methicillin-resistant Staphylococcus aureus (MRSA). Although the QacA and QacB proteins are similar to each other, their respective substrate specificities may differ. We investigated the variability and structure-function relationships of QacA and QacB in MRSA isolates. The amino acid sequences of 7 QacA and 25 QacB proteins showed that QacB was present in three variants, designated QacBII, QacBIII, and QacBIV, that were different from the prototypic QacB variant encoded by plasmid pSK23, which was named QacBI, while QacA was present in two variants. When cloned and expressed in S. aureus, the strain carrying qacBIII exhibited higher susceptibility to dyes and decreased susceptibility to norfloxacin and ciprofloxacin compared to strains carrying the other QacB variants. Site-directed mutagenesis experiments revealed that the residue at position 320 in QacB plays an important role in the resistance phenotypes to dyes and fluoroquinolones. Furthermore, the accumulation of norfloxacin and ciprofloxacin in the strain carrying qacBIII was significantly decreased. Our data demonstrate that the plasmid-mediated multidrug efflux pump QacB variant QacBIII confers the capability for fluoroquinolone efflux on S. aureus.

scholarly journals Covalently Linked Trimer of the AcrB Multidrug Efflux Pump Provides Support for the Functional Rotating Mechanism

2008 ◽  
Vol 191 (6) ◽  
pp. 1729-1737 ◽  
Author(s):  
Yumiko Takatsuka ◽  
Hiroshi Nikaido
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Efflux Pump ◽  
Polyacrylamide Gel Electrophoresis ◽  
Residual Activity ◽  
Salt Bridge ◽  
Relay Network ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Intact Cells

ABSTRACT Escherichia coli AcrB is a proton motive force-dependent multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export (functional rotation hypothesis). In this study, we devised a way to test this hypothesis by creating a giant gene in which three acrB sequences were connected together through short linker sequences. The “linked-trimer” AcrB was expressed well in the inner membrane fraction of ΔacrB ΔrecA strains, as a large protein of ∼300 kDa which migrated at the same rate as the wild-type AcrB trimer in native polyacrylamide gel electrophoresis. The strain expressing the linked-trimer AcrB showed resistance to some toxic compounds that was sometimes even higher than that of the cells expressing the monomeric AcrB, indicating that the linked trimer functions well in intact cells. When we inactivated only one of the three protomeric units in the linked trimer, either with mutations in the salt bridge/H-bonding network (proton relay network) in the transmembrane domain or by disulfide cross-linking of the external cleft in the periplasmic domain, the entire trimeric complex was inactivated. However, some residual activity was seen, presumably as a result of random recombination of monomeric fragments (produced by protease cleavage or by transcriptional/translational truncation). These observations provide strong biochemical evidence for the functionally rotating mechanism of AcrB pump action. The linked trimer will be useful for further biochemical studies of mechanisms of transport in the future.

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