Exploring Molecular Interactions between Escherichia coli RNA Polymerase and Topoisomerase I by Molecular Simulations

Tiwari, Purushottam; Chapagain, Prem; Banda, Srikanth; Darici, Yesim; Tse‐Dinh, Yuk‐Ching; Üren, Aykut

doi:10.1016/j.bpj.2016.11.1555

Cited by 3 publications

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“…Therefore, in vivo the enzyme should be able to relieve negative supercoiling generated by transcription and, possibly, replication, and compensate the DNA gyrase activity to maintain physiological level of supercoiling (Massé and Drolet, 1999;Zechiedrich et al, 2000). E. coli topoisomerase I was demonstrated to interact through its Znbinding and C-terminal domains with the β' subunit of the RNA polymerase (Cheng et al, 2003;Tiwari et al, 2017). Eukaryotic TOP1 and RNAPII, as well as mycobacterial and streptococcal TopoI and RNAP were also shown to interact.…”

Section: Discussionmentioning

confidence: 99%

“…We suggest that the Zn-binding domain of EcTopoI is primarily responsible for the binding to RNAP and/or its affinity to RNAP is sufficient for required levels of complex formation. Of note, molecular dynamics simulation predicted residues of Zn-binding domain, but not of CTD, to be involved in the interaction with RNAP (Tiwari et al, 2017). We assume that when CTD is overexpressed and binds to RNAP, it sterically excludes topoisomerase from the complex.…”

Section: Rnap:ectopoi Complex Is Required For R-loops Formation Controlmentioning

confidence: 98%

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Interaction Between Transcribing RNA Polymerase and Topoisomerase I Prevents R-loop Formation in E. coli

Sutormin

Galivondzhyan

Musharova

et al. 2021

Preprint

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Bacterial topoisomerase I (TopoI) removes excessive negative supercoiling and is thought to relax DNA molecules during transcription, replication and other processes. Using ChIP-Seq, we show that TopoI of Escherichia coli (EcTopoI) is co-localized, genome-wide, with RNA polymerase (RNAP) in transcription units. Treatment with transcription elongation inhibitor rifampicin leads to EcTopoI relocation to promoter regions, where RNAP also accumulates. When a 14 kDa RNAP-binding EcTopoI C-terminal domain (CTD) is overexpressed, co-localization of EcTopoI and RNAP along the transcription units is reduced. Pull-down experiments directly show that the two enzymes interact in vivo. Using ChIP-Seq and Topo-Seq, we demonstrate that EcTopoI is enriched and in and upstream (within up to 12-15 Kbs) of highly-active transcription units, indicating that EcTopoI relaxes negative supercoiling generated by transcription. Uncoupling of the RNAP-EcTopoI interaction by either overexpression of EcTopoI CTD or deletion of EcTopoI domains involved in the interaction is toxic for cells and leads to excessive negative plasmid supercoiling. Moreover, the CTD overexpression leads to R-loops accumulation genome-wide, indicating that the RNAP-EcTopoI interaction is required for prevention of R-loops formation.Article HighlightsTopoI colocalizes genome-wide and interacts with RNAP in E. coliDisruption of the interaction between TopoI and RNAP decreases cells viability, leads to hypernegative DNA supercoiling, and R-loops accumulationTopoI and DNA gyrase are enriched, respectively, upstream and downstream of transcription units in accordance with twin-domain model of Liu and WangTopoI recognizes its cleavage sites through a specific motif and by sensing negative supercoiling

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Section: Discussionmentioning

confidence: 99%

Section: Rnap:ectopoi Complex Is Required For R-loops Formation Controlmentioning

confidence: 98%

Interaction Between Transcribing RNA Polymerase and Topoisomerase I Prevents R-loop Formation in E. coli

Sutormin

Galivondzhyan

Musharova

et al. 2021

Preprint

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“…(Cheng et al, 2003). Analysis of MsmTopoI protein sequence (Bhaduri et al, 1998) and determination of D1-D6 structure of MtbTopoI (Tan et al, 2016) have Molecular simulations predicted that salt bridges and hydrogen bonds formed by basic residues positioned over a large molecular surface formed by the zinc ribbon motifs of E. coli TopoI are responsible for interactions with acidic residues in RNAP (Tiwari et al, 2016). In contrast, a novel mechanism utilizing a short stretch of C-terminal tail is employed instead for the TopoI-RNAP interaction in bacteria that do not have Zn (II) binding TopoI-CTD.…”

Section: Discussionmentioning

confidence: 99%

Protein-protein Interactions of Bacterial Topoisomerase I

Banda¹

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“…Overexpression of the recombinant 14 kDa C-terminal fragment (D8-D9) of TopA was toxic to the cell, probably due to the interaction of recombinant C-terminal fragment of TopA with the β' subunit of RNA polymerase abolishes the physiological interaction between RNA polymerase and full length TopA via its C-terminus domains D5-D9 (Cheng et al, 2003;Yang et al, 2015;Zhu et al, 1995). The structure-based modeling of the TopA and RNA polymerase interactions predicts amino acid residues Arg-609, Lys-627 and Lys-664 are involved in the formation and the stabilization of the TopA-RNAP complex (Tiwari et al, 2016).…”

Section: Chapter Three: Investigation Of Molecular Mechanism Of E Comentioning

confidence: 99%

Studies of Nε-Lysine Acetylation Modification on Escherichia coli Topoisomerase I

Zhou¹

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Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by N ε-Lysine acetylation. The sirtuin protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modifications. Here, we explored the effect of lysine acetylation on E. coli topoisomerase I through analysis of TopA relaxation activity and protein expression in cell extract of wild-type and a ΔcobB mutant strains. We showed that the absence of deacetylase CobB in a ΔcobB mutant reduced intracellular TopA relaxation activity while elevating TopA expression and topA gene transcripts levels. Acetyl phosphate mediated lysine acetylation decreased the activity of purified TopA in vitro, and the interaction with purified CobB protected TopA from such inactivation. We explored the physiological significance of TopA acetylation on DNA supercoiling by two-dimensional gel analysis and on cell growth rate by growth curve analysis. We found that the absence of CobB increased negative DNA supercoiling. The vii slow growth phenotype of the ∆cobB mutant can be partially compensated by overexpression of recombinant TopA. In addition, the specific activity of TopA expressed from His-tagged fusion construct in the chromosome was inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. Investigation of TopA relaxation mechanism using nuclease footprinting and TopA oxidative crosslinking suggested the potential association of TopA acetylation in catalysis. Mass spectrometry analysis of in vitro acetyl phosphate acetylated TopA identified abundant lysine acetylation sites. Substitution of lysine residues by site-directed mutagenesis was used to model the effect of acetylation on individual lysine residues. Our results showed that substitution of Lys-484 with alanine reduced the relaxation activity, suggesting the reduction of TopA relaxation activity by acetylation was probably in part due to acetylation on Lys-484. These findings demonstrate that E. coli topoisomerase I is modulated by lysine acetylation and the prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the sirtuin deacetylase CobB. viii

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Exploring Molecular Interactions between Escherichia coli RNA Polymerase and Topoisomerase I by Molecular Simulations

Cited by 3 publications

References 0 publications

Interaction Between Transcribing RNA Polymerase and Topoisomerase I Prevents R-loop Formation in E. coli

Interaction Between Transcribing RNA Polymerase and Topoisomerase I Prevents R-loop Formation in E. coli

Protein-protein Interactions of Bacterial Topoisomerase I

Studies of Nε-Lysine Acetylation Modification on Escherichia coli Topoisomerase I

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