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Kenneth J Mcdowall - One of the best experts on this subject based on the ideXlab platform.
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Erratum: ThE First Small-MolEculE Inhibitors of MEmbErs of thE RibonuclEasE E Family
Scientific Reports, 2015Co-Authors: Louise Kime, Helen A. Vincent, Deena M. A. Gendoo, Stefanie S. Jourdan, Colin W. G. Fishwick, Anastasia J. Callaghan, Kenneth J McdowallAbstract:ThE EschErichia coli EndoRibonuclEasE RNasE E is cEntral to thE procEssing and dEgradation of all typEs of RNA and as such is a plEotropic rEgulator of gEnE ExprEssion. It is EssEntial for growth and was onE of thE first ExamplEs of an EndonuclEasE that can rEcognisE thE 5′-monophosphorylatEd Ends of RNA thErEby incrEasing thE EfficiEncy of many clEavagEs. HomologuEs of RNasE E can bE found in many bactErial familiEs including important pathogEns, but no homologuEs havE bEEn idEntifiEd in humans or animals. RNasE E rEprEsEnts a potEntial targEt for thE dEvElopmEnt of nEw antibiotics to combat thE growing numbEr of bactEria that arE rEsistant to antibiotics in usE currEntly. PotEnt small molEculE inhibitors that bind thE activE sitE of EssEntial EnzymEs arE proving to bE a sourcE of potEntial drug lEads and tools to dissEct function through chEmical gEnEtics. HErE wE rEport thE usE of virtual high-throughput scrEEning to obtain small molEculEs prEdictEd to bind at sitEs in thE N-tErminal catalytic half of RNasE E. WE show that thEsE compounds arE ablE to bind with spEcificity and inhibit catalysis of EschErichia coli and MycobactErium tubErculosis RNasE E and also inhibit thE activity of RNasE G, a paraloguE of RNasE E.
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ThE First Small-MolEculE Inhibitors of MEmbErs of thE RibonuclEasE E Family
Scientific Reports, 2015Co-Authors: Louise Kime, Helen A. Vincent, Deena M. A. Gendoo, Stefanie S. Jourdan, Colin W. G. Fishwick, Anastasia J. Callaghan, Kenneth J McdowallAbstract:ThE EschErichia coli EndoRibonuclEasE RNasE E is cEntral to thE procEssing and dEgradation of all typEs of RNA and as such is a plEotropic rEgulator of gEnE ExprEssion. It is EssEntial for growth and was onE of thE first ExamplEs of an EndonuclEasE that can rEcognisE thE 5′-monophosphorylatEd Ends of RNA thErEby incrEasing thE EfficiEncy of many clEavagEs. HomologuEs of RNasE E can bE found in many bactErial familiEs including important pathogEns, but no homologuEs havE bEEn idEntifiEd in humans or animals. RNasE E rEprEsEnts a potEntial targEt for thE dEvElopmEnt of nEw antibiotics to combat thE growing numbEr of bactEria that arE rEsistant to antibiotics in usE currEntly. PotEnt small molEculE inhibitors that bind thE activE sitE of EssEntial EnzymEs arE proving to bE a sourcE of potEntial drug lEads and tools to dissEct function through chEmical gEnEtics. HErE wE rEport thE usE of virtual high-throughput scrEEning to obtain small molEculEs prEdictEd to bind at sitEs in thE N-tErminal catalytic half of RNasE E. WE show that thEsE compounds arE ablE to bind with spEcificity and inhibit catalysis of EschErichia coli and MycobactErium tubErculosis RNasE E and also inhibit thE activity of RNasE G, a paraloguE of RNasE E.
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“Zn-Link”: a mEtal-sharing intErfacE that organizEs thE quatErnary structurE and catalytic sitE of thE EndoRibonuclEasE, RNasE E
Biochemistry, 2005Co-Authors: Anastasia J. Callaghan, Yulia U Redko, Carol V Robinson, Leopold L Ilag, Martyn F Symmons, L.m. Murphy, J.g. Grossmann, David Yates, Elspeth F. Garman, Kenneth J McdowallAbstract:RibonuclEasE E is an EssEntial hydrolytic EndonuclEasE in EschErichia coli, and it plays a cEntral rolE in maintaining thE balancE and composition of thE mEssEngEr RNA population. ThE EnzymE is also rEquirEd for rRNA and tRNA procEssing. WE havE shown EarliEr that thE highly consErvEd catalytic domain of E. coli RNasE E is a homotEtramEr [Callaghan, A. J. Et al. (2003) BiochEmistry 42, 13848−13855]. HErE, wE rEport that this quatErnary organization rEquirEs zinc. Two protomErs sharE a singlE zinc ion, and quantitativE analysis indicatEs that Each protEin contributEs two cystEinE thiols toward thE coordination of thE mEtal. ThE candidatE cystEinEs arE part of a motif that is consErvEd in thE RNasE E protEin family, and mutation of thEsE rEsiduEs causEs thE partial loss of zinc, thE complEtE disruption of thE tEtramEr into dimErs, and EffEctivE catalytic inactivation. HowEvEr, thEsE mutations do not affEct RNA binding. ThE tEtramEr can bE artificially maintainEd by disulfidE bond formation, which fully disp...
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dEtErmination of thE catalytic paramEtErs of thE n tErminal half of EschErichia coli RibonuclEasE E and thE idEntification of critical functional groups in rna substratEs
Journal of Biological Chemistry, 2003Co-Authors: Yulia U Redko, Mark R Tock, Chris J Adams, Vladimir R Kaberdin, Jane A Grasby, Kenneth J McdowallAbstract:RibonuclEasE E is rEquirEd for thE rapid dEcay and corrEct procEssing of RNA in EschErichia coli. A dEtailEd undErstanding of thE hydrolysis of RNA by this and rElatEd EnzymEs will rEquirE thE intEgration of structural and molEcular data with quantitativE mEasurEmEnts of RNA hydrolysis. ThErEforE, an assay for RNasEE that can bE sEt up to havE rElativEly high throughput whilE bEing sEnsitivE and quantitativE will bE advantagEous. HErE wE dEscribE such an assay, which is basEd on thE automatEd high prEssurE liquid chromatography analysis of fluorEscEntly labElEd RNA samplEs. WE havE usEd this assay to optimizE rEaction conditions, to dEtErminE for thE first timE thE catalytic paramEtErs for a polypEptidE of RNasEE, and to invEstigatE thE RNasEE-catalyzEd rEaction through thE modification of functional groups within an RNA substratE. WE find that catalysis is dEpEndEnt on both protonatEd and unprotonatEd functional groups and that thE rEcognition of a guanosinE sEquEncE dEtErminant that is upstrEam of thE scissilE bond appEars to consist of intEractions with thE Exocyclic 2-amino group, thE 7N of thE nuclEobasE and thE imino proton or 6-kEto group. Additionally, wE find that a ribosE-likE sugar conformation is prEfErrEd in thE 5'-nuclEotidE of thE scissilE phosphodiEstEr bond and that a 2'-hydroxyl group proton is not EssEntial. StEric bulk at thE 2' position in thE 5'-nuclEotidE appEars to bE inhibitory to thE rEaction. CombinEd, thEsE obsErvations Establish a foundation for thE functional intErprEtation of a thrEE-dimEnsional structurE of thE catalytic domain of RNasEE whEn solvEd.
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quatErnary structurE and catalytic activity of thE EschErichia coli RibonuclEasE E amino tErminal catalytic domain
Biochemistry, 2003Co-Authors: Anastasia J. Callaghan, Yulia U Redko, Kenneth J Mcdowall, Carol V Robinson, Gunter J Grossmann, Leopold L Ilag, Martin C Moncrieffe, Martyn F Symmons, Ben F LuisiAbstract:RNasE E is an EssEntial EndoRibonuclEasE that plays a cEntral rolE in thE procEssing and dEgradation of RNA in EschErichia coli and othEr bactEria. Most EndoRibonuclEasEs havE bEEn shown to act distributivEly; howEvEr, FEng Et al. [(2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14746−14751] havE rEcEntly found that RNasE E acts via a scanning mEchanism. A structural Explanation for thE procEssivity of RNasE E is providEd hErE, with our finding that thE consErvEd catalytic domain of E. coli RNasE E forms a homotEtramEr. Nondissociating nanoflow-ElEctrospray mass spEctromEtry suggEsts that thE tEtramEr binds up to four molEculEs of a spEcific substratE RNA analoguE. ThE tEtramEric assEmbly of thE N-tErminal domain of RNasE E is consistEnt with crystallographic analysEs, which indicatE that thE tEtramEr possEssEs approximatE D2 dihEdral symmEtry. Using X-ray solution scattEring data and symmEtry rEstraints, a solution shapE is calculatEd for thE tEtramEr. This shapE, togEthEr with limitEd protEolysis data, suggEst...
Stanley N Cohen - One of the best experts on this subject based on the ideXlab platform.
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RibonuclEasE E modulation of thE bactErial sos rEsponsE
PLOS ONE, 2012Co-Authors: Robert Manasherob, Christine A Miller, Stanley N CohenAbstract:Plants, animals, bactEria, and ArchaEa all havE EvolvEd mEchanisms to copE with EnvironmEntal or cEllular strEss. BactErial cElls rEspond to thE strEss of DNA damagE by activation of thE SOS rEsponsE, thE canonical REcA/LExA-dEpEndEnt signal transduction pathway that transcriptionally dErEprEssEs a multiplicity of gEnEs–lEading to transiEnt arrEst of cEll division and initiation of DNA rEpair. HErE wE rEport thE prEviously unsuspEctEd rolE of E. coli EndoRibonuclEasE RNasE E in rEgulation of thE SOS rEsponsE. WE show that RNasE E dElEtion or inactivation of tEmpEraturE-sEnsitivE RNasE E protEin prEcludEs normal initiation of SOS. ThE ability of RNasE E to rEgulatE SOS is dynamic, as down rEgulation of RNasE E following DNA damagE by mitomycin C rEsultEd in SOS tErmination and rEstoration of RNasE E function lEads to rEsumption of a prEviously abortEd rEsponsE. OvErExprEssion of thE RraA protEin, which binds to thE C-tErminal rEgion of RNasE E and modulatEs thE actions of dEgradosomEs, rEcapitulatEd thE EffEcts of RNasE E dEficiEncy. PossiblE mEchanisms for RNasE E EffEcts on SOS arE discussEd.
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rEtEntion of corE catalytic functions by a consErvEd minimal RibonuclEasE E pEptidE that lacks thE domain rEquirEd for tEtramEr formation
Journal of Biological Chemistry, 2006Co-Authors: Jonathan M Caruthers, Yanan Feng, David B Mckay, Stanley N CohenAbstract:Abstract RibonuclEasE E (RNasE E) is a multifunctional EndoRibonuclEasE that has bEEn Evolutionarily consErvEd in both Gram-positivE and Gram-nEgativE bactEria. X-ray crystallography and biochEmical studiEs havE concludEd that thE EschErichia coli RNasE E protEin functions as a homotEtramEr formEd by Zn linkagE of dimErs within a rEgion ExtEnding from amino acid rEsiduEs 416 through 529 of thE 116-kDa protEin. Using fragmEnts of RNasE E protEins from E. coli and HaEmophilus influEnzaE, wE show hErE that RNasE E dErivativEs that arE as short as 395 amino acid rEsiduEs and that lack thE Zn-link rEgion shown prEviously to bE EssEntial for tEtramEr formation (i.E. amino acid rEsiduEs 400–415) arE catalytically activE EnzymEs that rEtain thE 5′ to 3′ scanning ability and clEavagE sitE spEcificity charactEristic of full-lEngth RNasE E and that also confEr colony forming ability on rnE null mutant bactEria. FurthEr truncation lEads to loss of thEsE propErtiEs. Our rEsults, which idEntify a minimal catalytically activE RNasE E sEquEncE, indicatE that contrary to currEnt modEls, a tEtramEric quatErnary structurE is not rEquirEd for RNasE E to carry out its corE Enzymatic functions.
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RraA: a ProtEin Inhibitor of RNasE E Activity that Globally ModulatEs RNA AbundancE in E. coli
Cell, 2003Co-Authors: Xiaoming Zhan, Yanan Feng, Stanley N Cohen, R. Meganathan, George GeorgiouAbstract:Abstract RibonuclEasE E (RNasE E) has a kEy rolE in mRNA dEgradation and thE procEssing of catalytic and structural RNAs in E. coli . WE rEport thE discovEry of an Evolutionarily consErvEd 17.4 kDa protEin, hErE namEd RraA ( r Egulator of r ibonuclEasE a ctivity A) that binds to RNasE E and inhibits RNasE E EndonuclEolytic clEavagEs without altEring clEavagE sitE spEcificity or intEracting dEtEctably with substratE RNAs. OvErExprEssion of RraA circumvEnts thE EffEcts of an autorEgulatory mEchanism that normally maintains thE RNasE E cEllular lEvEl within a narrow rangE, rEsulting in thE gEnomE-widE accumulation of RNasE E-targEtEd transcripts. WhilE not rEquirEd for RraA action, thE C-tErminal RNasE E rEgion that sErvEs as a scaffold for formation of a multiprotEin dEgradosomE complEx modulatEs thE inhibition of RNasE E catalytic activity by RraA. Our rEsults rEvEal a possiblE mEchanism for thE dynamic rEgulation of RNA dEcay and procEssing by inhibitory RNasE binding protEins.
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rna dEgradosomEs Exist in vivo in EschErichia coli as multicomponEnt complExEs associatEd with thE cytoplasmic mEmbranE via thE n tErminal rEgion of RibonuclEasE E
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Gunnguang Liou, Stanley N Cohen, Wannneng Jane, Sue LinchaoAbstract:RNasE E isolatEd from EschErichia coli is containEd in a multicomponEnt “dEgradosomE” complEx with othEr protEins implicatEd in RNA dEcay. EarliEr work has shown that thE C-tErminal rEgion of RNasE E is a scaffold for thE binding of dEgradosomE componEnts and has idEntifiEd spEcific RNasE E sEgmEnts nEcEssary for its intEraction with polynuclEotidE phosphorylasE (PNPasE), RhlB RNA hElicasE, and EnolasE. HErE, wE rEport ElEctron microscopy studiEs that usE immunogold labEling and frEEzE–fracturE mEthods to show that dEgradosomEs Exist in vivo in E. coli as multicomponEnt structurEs that associatE with thE cytoplasmic mEmbranE via thE N-tErminal rEgion of RNasE E. WhErEas PNPasE and EnolasE arE prEsEnt in E. coli in largE ExcEss rElativE to RNasE E and thErEforE arE dEtEctEd in cElls largEly as molEculEs unlinkEd to thE RNasE E scaffold, immunogold labEling and biochEmical analysEs show that hElicasE is prEsEnt in approximatEly Equimolar amounts to RNasE E at all cEll growth stagEs. Our findings, which Establish thE ExistEncE and cEllular location of RNasE E-basEd dEgradosomEs in vivo in E. coli, also suggEst that RNA procEssing and dEcay may occur at spEcific sitEs within cElls.
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AltErnativE splicing rEgulatEs thE production of ARD-1 EndoRibonuclEasE and NIPP-1, an inhibitor of protEin phosphatasE-1, as isoforms EncodEd by thE samE gEnE.
Gene, 1999Co-Authors: Annie C. Y. Chang, Björn Sohlberg, Laura Trinkle-mulcahy, Felix Claverie-martin, Philip Cohen, Stanley N CohenAbstract:Abstract ARD-1 is an EndoRibonuclEasE idEntifiEd initially as thE product of a human cDNA that complEmEnts mutations in rnE, a gEnE that EncodEs EschErichia coli RibonuclEasE E. NIPP-1 was idEntifiEd in bovinE nuclEar Extracts as an inhibitor of protEin phosphatasE-1. EarliEr work has shown that thE protEin-coding sEquEncE of ARD-1 is idEntical to thE carboxy-tErminal third of NIPP-1. HowEvEr, whEthEr ARD-1 is prEsEnt in EukaryotEs as a distinct Entity has bEEn unclEar, as nEithEr ARD-1-spEcific transcripts nor ARD-1 protEin wErE dEtEctEd in mammalian cElls in EarliEr studiEs. HErE wE show that ARD-1 Exists in human cElls as a discrEtE protEin, and that thE ARD-1 and NIPP-1 pEptidEs arE isoforms EncodEd by a singlE gEnE and thE samE altErnativEly splicEd prEcursor RNA. A rEtainEd intron containing multiplE translation stop codons that arE configurEd to tErminatE translation and initiatE nonsEnsE-mEdiatEd dEcay, limits thE production of cEllular ARD-1 protEin. Our rEsults Establish thE procEss by which functionally disparatE ARD-1 and NIPP-1 pEptidEs arE gEnEratEd from thE protEin-coding sEquEncE of thE samE gEnE in human cElls.
Vladimir R Kaberdin - One of the best experts on this subject based on the ideXlab platform.
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rEgulation of RibonuclEasE E activity by thE l4 ribosomal protEin of EschErichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Dharam Singh, Vladimir R Kaberdin, Ssujean Chang, Olga V Averina, Sue LinchaoAbstract:WhErEas ribosomal protEins (r-protEins) arE known primarily as componEnts of thE translational machinEry, cErtain of thEsE r-protEins havE bEEn found to also havE Extraribosomal functions. HErE wE rEport thE novEl ability of an r-protEin, L4, to rEgulatE RNA dEgradation in EschErichia coli. WE show by affinity purification, immunoprEcipitation analysis, and E. coli two-hybrid scrEEning that L4 intEracts with a sitE outsidE of thE catalytic domain of RNasE E to rEgulatE thE EndoribonuclEolytic functions of thE EnzymE, thus inhibiting RNasE E-spEcific clEavagE in vitro, stabilizing mRNAs targEtEd by RNasE E in vivo, and controlling plasmid DNA rEplication by stabilizing an antisEnsE rEgulatory RNA normally attackEd by RNasE E. BroadEr EffEcts of thE L4-RNasE E intEraction on E. coli transcripts wErE shown by DNA microarray analysis, which rEvEalEd changEs in thE abundancE of 65 mRNAs Encoding thE strEss rEsponsE protEins HslO, Lon, CstA, YjiY, and YaEL, as wEll as protEins involvEd in carbohydratE and amino acid mEtabolism and transport, transcription/translation, and DNA/RNA synthEsis. Analysis of mRNA stability showEd that thE half livEs of strEss-rEsponsivE transcripts wErE incrEasEd by Ectopic ExprEssion of L4, which normally incrEasEs along with othEr r-protEins in E. coli undEr strEss conditions, and also by inactivation of RNasE E. Our finding that L4 can inhibit RNasE E-dEpEndEnt dEcay may account at lEast in part for thE ElEvatEd production of strEss-inducEd protEins during bactErial adaptation to advErsE EnvironmEnts.
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dEtErmination of thE catalytic paramEtErs of thE n tErminal half of EschErichia coli RibonuclEasE E and thE idEntification of critical functional groups in rna substratEs
Journal of Biological Chemistry, 2003Co-Authors: Yulia U Redko, Mark R Tock, Chris J Adams, Vladimir R Kaberdin, Jane A Grasby, Kenneth J McdowallAbstract:RibonuclEasE E is rEquirEd for thE rapid dEcay and corrEct procEssing of RNA in EschErichia coli. A dEtailEd undErstanding of thE hydrolysis of RNA by this and rElatEd EnzymEs will rEquirE thE intEgration of structural and molEcular data with quantitativE mEasurEmEnts of RNA hydrolysis. ThErEforE, an assay for RNasEE that can bE sEt up to havE rElativEly high throughput whilE bEing sEnsitivE and quantitativE will bE advantagEous. HErE wE dEscribE such an assay, which is basEd on thE automatEd high prEssurE liquid chromatography analysis of fluorEscEntly labElEd RNA samplEs. WE havE usEd this assay to optimizE rEaction conditions, to dEtErminE for thE first timE thE catalytic paramEtErs for a polypEptidE of RNasEE, and to invEstigatE thE RNasEE-catalyzEd rEaction through thE modification of functional groups within an RNA substratE. WE find that catalysis is dEpEndEnt on both protonatEd and unprotonatEd functional groups and that thE rEcognition of a guanosinE sEquEncE dEtErminant that is upstrEam of thE scissilE bond appEars to consist of intEractions with thE Exocyclic 2-amino group, thE 7N of thE nuclEobasE and thE imino proton or 6-kEto group. Additionally, wE find that a ribosE-likE sugar conformation is prEfErrEd in thE 5'-nuclEotidE of thE scissilE phosphodiEstEr bond and that a 2'-hydroxyl group proton is not EssEntial. StEric bulk at thE 2' position in thE 5'-nuclEotidE appEars to bE inhibitory to thE rEaction. CombinEd, thEsE obsErvations Establish a foundation for thE functional intErprEtation of a thrEE-dimEnsional structurE of thE catalytic domain of RNasEE whEn solvEd.
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clEavagE of poly a tails on thE 3 End of rna by RibonuclEasE E of EschErichia coli
Nucleic Acids Research, 2001Co-Authors: Andrew P Walsh, Mark R Tock, Vladimir R Kaberdin, M H Mallen, A Von Gabain, Kenneth J McdowallAbstract:RNasE E initiatEs thE dEcay of EschErichia coli RNAs by cutting thEm intErnally nEar thEir 5′-End and is a componEnt of thE RNA dEgradosomE complEx, which also contains thE 3′-ExonuclEasE PNPasE. REcEntly, RNasE E has bEEn shown to bE ablE to rEmovE poly(A) tails by what has bEEn dEscribEd as an ExonuclEolytic procEss that can bE blockEd by thE prEsEncE of a phosphatE group on thE 3′-End of thE RNA. WE show hErE, howEvEr, that poly(A) tail rEmoval by RNasE E is in fact an EndonuclEolytic procEss that is rEgulatEd by thE phosphorylation status at thE 5′- but not thE 3′-End of RNA. ThE ratE of poly(A) tail rEmoval by RNasE E was found to bE 30-fold grEatEr whEn thE 5′-tErminus of RNA substratEs was convErtEd from a triphosphatE to monophosphatE group. This finding promptEd us to rE-analysE thE contributions of thE ribonuclEolytic activitiEs within thE dEgradosomE to 3′ attack sincE prEvious studiEs had only usEd substratEs that had a triphosphatE group on thEir 5′-End. Our rEsults indicatE that RNasE E associatEd with thE dEgradosomE may contributE to thE rEmoval of poly(A) tails from 5′-monophosphorylatEd RNAs, but this is only likEly to bE significant should thEir attack by PNPasE bE blockEd.
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EnhancEd clEavagE of rna mEdiatEd by an intEraction bEtwEEn substratEs and thE argininE rich domain of E coli RibonuclEasE E
Journal of Molecular Biology, 2000Co-Authors: Vladimir R Kaberdin, Kenneth J Mcdowall, Andrew P Walsh, Thania Jakobsen, Alexander Von GabainAbstract:Abstract EndonuclEolytic cutting by thE EssEntial EschErichia coli RibonuclEasE RNasEE has a cEntral rolE in both thE procEssing and dEcay of RNA. PrEviously, it has bEEn shown that an oligoribonuclEotidE corrEsponding in sEquEncE to thE singlE-strandEd rEgion at thE 5′ End of RNAI, thE antisEnsE rEgulator of ColE1-typE plasmid rEplication, is EfficiEntly cut by RNasEE. CombinEd with thE knowlEdgE that altEration of thE structurE of stEm-loops within complEx RNasEE substratEs can EithEr incrEasE or dEcrEasE thE ratE of clEavagE, this rEsult has lEd to thE notion that stEm-loops do not sErvE as EssEntial rEcognition motifs for RNasEE, but can affEct thE ratE of clEavagE indirEctly by, for ExamplE, dEtErmining thE singlE-strandEdnEss of thE sitE or its accEssibility. WE rEport hErE, howEvEr, that not all oligoribonuclEotidEs corrEsponding to RNasEE-clEavEd sEgmEnts of complEx substratEs arE sufficiEnt to dirEct EfficiEnt RNasEE clEavagE. WE providE EvidEncE using 9 S RNA, a prEcursor of 5 S rRNA, that binding of structurEd rEgions by thE argininE-rich RNA- binding domain (ARRBD) of RNasEE can bE rEquirEd for EfficiEnt clEavagE. Binding by thE ARRBD appEars to countEract thE inhibitory EffEcts of sub-optimal clEavagE sitE sEquEncE and ovErall substratE conformation. FurthErmorE, combinEd with thE rEsults from rEcEnt analysEs of E. coli mutants in which thE ARRBD of RNasE E is dElEtEd, our findings suggEst that substratE binding by RNasEE is EssEntial for thE normal rapid dEcay of E. coli mRNA. ThE simplEst intErprEtation of our rEsults is that thE ARRBD rEcruits RNasEE to structurEd RNAs, thErEby incrEasing thE localisEd concEntration of thE N-tErminal catalytic domain, which in turn lEads to an incrEasE in thE ratE of clEavagE.
Anastasia J. Callaghan - One of the best experts on this subject based on the ideXlab platform.
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A structural and biochEmical comparison of RibonuclEasE E homologuEs from pathogEnic bactEria highlights spEciEs-spEcific propErtiEs
Scientific Reports, 2019Co-Authors: Charlotte E. Mardle, Helen A. Vincent, Thomas J. Shakespeare, Louise E. Butt, Layla R. Goddard, Darren M. Gowers, Helen S. Atkins, Anastasia J. CallaghanAbstract:REgulation of gEnE ExprEssion through procEssing and turnovEr of RNA is a kEy mEchanism that allows bactEria to rapidly adapt to changing EnvironmEntal conditions. ConsEquEntly, RNA dEgrading EnzymEs (RibonuclEasEs; RNasEs) such as thE EndoRibonuclEasE RNasE E, frEquEntly play critical rolEs in pathogEnic bactErial virulEncE and arE potEntial antibactErial targEts. RNasE E consists of a highly consErvEd catalytic domain and a variablE non-catalytic domain that functions as thE structural scaffold for thE multiEnzymE dEgradosomE complEx. DEspitE consErvation of thE catalytic domain, a rEcEnt study idEntifiEd diffErEncEs in thE rEsponsE of RNasE E homologuEs from diffErEnt spEciEs to thE samE inhibitory compound(s). WhilE RNasE E from EschErichia coli has bEEn wEll-charactErisEd, far lEss is known about RNasE E homologuEs from othEr bactErial spEciEs. In this study, wE structurally and biochEmically charactErisE thE RNasE E catalytic domains from four pathogEnic bactEria: YErsinia pEstis , FrancisElla tularEnsis , BurkholdEria psEudomallEi and AcinEtobactEr baumannii , with a viEw to Exploiting RNasE E as an antibactErial targEt. Bioinformatics, small-anglE x-ray scattEring and biochEmical RNA clEavagE assays rEvEal globally similar structural and catalytic propErtiEs. Surprisingly, subtlE spEciEs-spEcific diffErEncEs in both structurE and substratE spEcificity wErE also idEntifiEd that may bE important for thE dEvElopmEnt of EffEctivE antibactErial drugs targEting RNasE E.
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Erratum: ThE First Small-MolEculE Inhibitors of MEmbErs of thE RibonuclEasE E Family
Scientific Reports, 2015Co-Authors: Louise Kime, Helen A. Vincent, Deena M. A. Gendoo, Stefanie S. Jourdan, Colin W. G. Fishwick, Anastasia J. Callaghan, Kenneth J McdowallAbstract:ThE EschErichia coli EndoRibonuclEasE RNasE E is cEntral to thE procEssing and dEgradation of all typEs of RNA and as such is a plEotropic rEgulator of gEnE ExprEssion. It is EssEntial for growth and was onE of thE first ExamplEs of an EndonuclEasE that can rEcognisE thE 5′-monophosphorylatEd Ends of RNA thErEby incrEasing thE EfficiEncy of many clEavagEs. HomologuEs of RNasE E can bE found in many bactErial familiEs including important pathogEns, but no homologuEs havE bEEn idEntifiEd in humans or animals. RNasE E rEprEsEnts a potEntial targEt for thE dEvElopmEnt of nEw antibiotics to combat thE growing numbEr of bactEria that arE rEsistant to antibiotics in usE currEntly. PotEnt small molEculE inhibitors that bind thE activE sitE of EssEntial EnzymEs arE proving to bE a sourcE of potEntial drug lEads and tools to dissEct function through chEmical gEnEtics. HErE wE rEport thE usE of virtual high-throughput scrEEning to obtain small molEculEs prEdictEd to bind at sitEs in thE N-tErminal catalytic half of RNasE E. WE show that thEsE compounds arE ablE to bind with spEcificity and inhibit catalysis of EschErichia coli and MycobactErium tubErculosis RNasE E and also inhibit thE activity of RNasE G, a paraloguE of RNasE E.
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ThE First Small-MolEculE Inhibitors of MEmbErs of thE RibonuclEasE E Family
Scientific Reports, 2015Co-Authors: Louise Kime, Helen A. Vincent, Deena M. A. Gendoo, Stefanie S. Jourdan, Colin W. G. Fishwick, Anastasia J. Callaghan, Kenneth J McdowallAbstract:ThE EschErichia coli EndoRibonuclEasE RNasE E is cEntral to thE procEssing and dEgradation of all typEs of RNA and as such is a plEotropic rEgulator of gEnE ExprEssion. It is EssEntial for growth and was onE of thE first ExamplEs of an EndonuclEasE that can rEcognisE thE 5′-monophosphorylatEd Ends of RNA thErEby incrEasing thE EfficiEncy of many clEavagEs. HomologuEs of RNasE E can bE found in many bactErial familiEs including important pathogEns, but no homologuEs havE bEEn idEntifiEd in humans or animals. RNasE E rEprEsEnts a potEntial targEt for thE dEvElopmEnt of nEw antibiotics to combat thE growing numbEr of bactEria that arE rEsistant to antibiotics in usE currEntly. PotEnt small molEculE inhibitors that bind thE activE sitE of EssEntial EnzymEs arE proving to bE a sourcE of potEntial drug lEads and tools to dissEct function through chEmical gEnEtics. HErE wE rEport thE usE of virtual high-throughput scrEEning to obtain small molEculEs prEdictEd to bind at sitEs in thE N-tErminal catalytic half of RNasE E. WE show that thEsE compounds arE ablE to bind with spEcificity and inhibit catalysis of EschErichia coli and MycobactErium tubErculosis RNasE E and also inhibit thE activity of RNasE G, a paraloguE of RNasE E.
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“Zn-Link”: a mEtal-sharing intErfacE that organizEs thE quatErnary structurE and catalytic sitE of thE EndoRibonuclEasE, RNasE E
Biochemistry, 2005Co-Authors: Anastasia J. Callaghan, Yulia U Redko, Carol V Robinson, Leopold L Ilag, Martyn F Symmons, L.m. Murphy, J.g. Grossmann, David Yates, Elspeth F. Garman, Kenneth J McdowallAbstract:RibonuclEasE E is an EssEntial hydrolytic EndonuclEasE in EschErichia coli, and it plays a cEntral rolE in maintaining thE balancE and composition of thE mEssEngEr RNA population. ThE EnzymE is also rEquirEd for rRNA and tRNA procEssing. WE havE shown EarliEr that thE highly consErvEd catalytic domain of E. coli RNasE E is a homotEtramEr [Callaghan, A. J. Et al. (2003) BiochEmistry 42, 13848−13855]. HErE, wE rEport that this quatErnary organization rEquirEs zinc. Two protomErs sharE a singlE zinc ion, and quantitativE analysis indicatEs that Each protEin contributEs two cystEinE thiols toward thE coordination of thE mEtal. ThE candidatE cystEinEs arE part of a motif that is consErvEd in thE RNasE E protEin family, and mutation of thEsE rEsiduEs causEs thE partial loss of zinc, thE complEtE disruption of thE tEtramEr into dimErs, and EffEctivE catalytic inactivation. HowEvEr, thEsE mutations do not affEct RNA binding. ThE tEtramEr can bE artificially maintainEd by disulfidE bond formation, which fully disp...
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quatErnary structurE and catalytic activity of thE EschErichia coli RibonuclEasE E amino tErminal catalytic domain
Biochemistry, 2003Co-Authors: Anastasia J. Callaghan, Yulia U Redko, Kenneth J Mcdowall, Carol V Robinson, Gunter J Grossmann, Leopold L Ilag, Martin C Moncrieffe, Martyn F Symmons, Ben F LuisiAbstract:RNasE E is an EssEntial EndoRibonuclEasE that plays a cEntral rolE in thE procEssing and dEgradation of RNA in EschErichia coli and othEr bactEria. Most EndoRibonuclEasEs havE bEEn shown to act distributivEly; howEvEr, FEng Et al. [(2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14746−14751] havE rEcEntly found that RNasE E acts via a scanning mEchanism. A structural Explanation for thE procEssivity of RNasE E is providEd hErE, with our finding that thE consErvEd catalytic domain of E. coli RNasE E forms a homotEtramEr. Nondissociating nanoflow-ElEctrospray mass spEctromEtry suggEsts that thE tEtramEr binds up to four molEculEs of a spEcific substratE RNA analoguE. ThE tEtramEric assEmbly of thE N-tErminal domain of RNasE E is consistEnt with crystallographic analysEs, which indicatE that thE tEtramEr possEssEs approximatE D2 dihEdral symmEtry. Using X-ray solution scattEring data and symmEtry rEstraints, a solution shapE is calculatEd for thE tEtramEr. This shapE, togEthEr with limitEd protEolysis data, suggEst...
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RibonuclEasE p procEssEs polycistronic trna transcripts in EschErichia coli indEpEndEnt of RibonuclEasE E
Nucleic Acids Research, 2007Co-Authors: Bijoy K Mohanty, Sidney R KushnerAbstract:ThE first stEp in thE currEnt modEl for thE procEssing and maturation of mono- and polycistronic tRNA prEcursors in EschErichia coli involvEs initial clEavagEs by RNasE E 1–3nt downstrEam of Each chromosomally EncodEd CCA dEtErminant. SubsEquEntly, Each maturE 5’ tErminus is gEnEratEd by singlE RNasE P clEavagE, whilE thE 3’ tErminus undErgoEs ExonuclEolytic procEssing by a combination of 3’ !5’ ExonuclEasEs. HErE wE dEscribE for thE first timE a prEviously unidEntifiEd pathway for thE maturation of tRNAs in polycistronic opErons (valV valW and lEuQ lEuP lEuV) whErE thE procEssing of thE primary transcripts is indEpEndEnt of RNasE E. RathEr, RNasE P clEavagEs sEparatE thE individual tRNA prEcursors with thE concomitant formation of thEir maturE 5’ tErmini. FurthErmorE, both polynuclEotidE phosphorylasE (PNPasE) and RNasE II arE rEquirEd for thE rEmoval of thE 3’ RhodEpEndEnt tErminator sEquEncEs. Our data indicatE that RNasE P substratE rEcognition is morE complEx than prEviously EnvisionEd.
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Initiation of tRNA maturation by RNasE E is EssEntial for cEll viability in E. coli.
Genes & Development, 2002Co-Authors: Maria C. Ow, Sidney R KushnerAbstract:RibonuclEasE E (RNasE E) of EschErichia coli was first charactErizEd in a tEmpEraturE-sEnsitivE mutant (rnE-3071) that accumulatEd 9S prEcursors of thE 5S rRNA at 42°C (Ghora and Apirion 1978). IndEpEndEntly, thE ams (altErEd mRNA stability) gEnE was idEntifiEd bEcausE of its ability to affEct thE dEcay of total pulsE-labElEd RNA at ElEvatEd tEmpEraturEs (Ono and Kuwano 1979). SubsEquEntly, both loci wErE shown to EncodE RNasE E (Mudd Et al. 1990; BabitzkE and KushnEr 1991; TarasEviciEnE Et al. 1991). ThE rnE gEnE EncodEs a 1061-amino-acid protEin (CasarEgola Et al. 1992, 1994) that has now bEEn charactErizEd as a 5′-End-dEpEndEnt EndoRibonuclEasE (MackiE 1998). In addition, it has also bEEn shown that RNasE E is part of a multiprotEin complEx, callEd thE dEgradosomE, that includEs polynuclEotidE phosphorylasE (PNPasE), thE RhlB RNA hElicasE, and thE glycolytic EnzymE EnolasE (Carpousis Et al. 1994; Py Et al. 1994, 1996; Miczak 1996). In vivo ExpErimEnts with EithEr thE rnE-1 or rnE-3071 tEmpEraturE-sEnsitivE allElEs havE shown thE accumulation of unprocEssEd 5S rRNA intErmEdiatEs (Ghora and Apirion 1978; BabitzkE Et al. 1993) and a gEnEral slowing in thE dEcay of spEcific mRNA transcripts (Arraiano Et al. 1988; MackiE 1991; REgniEr and Hajnsdorf 1991). As such it was assumEd that thE inviability associatEd with thE inactivation of RNasE E rEsultEd from a dEfEct in EithEr 9S rRNA procEssing or mRNA dEcay. HowEvEr, ExpErimEnts by LopEz Et al. (1999) and Ow Et al. (2000) suggEst that this hypothEsis is not corrEct. For ExamplE, using rnE dElEtion mutations, both laboratoriEs showEd that 9S rRNA procEssing was almost normal undEr conditions in which mRNA dEcay was significantly impairEd (LopEz Et al. 1999; Ow Et al. 2000). FurthErmorE, Ow Et al. (2000) charactErizEd an ExtEnsivE RNasE E C tErminus truncation mutation (rnEΔ610) that was missing 609 amino acids, including thE ARRBS (argininE-rich RNA-binding sitE) and thE dEgradosomE scaffolding rEgion (Fig. (Fig.1).1). This protEin was ablE to support cEll viability at 37°C but not at 44°C. MorE importantly, dEcay of spEcific mRNAs was morE dEfEctivE at 37°C than in an rnE-1 mutant shiftEd to thE nonpErmissivE tEmpEraturE. ThErEforE, it was concludEd that inviability in thE absEncE of RNasE E was not associatEd with dEfEcts in EithEr mRNA dEcay or 9S rRNA procEssing (Ow Et al. 2000). FigurE 1 SchEmatic rEprEsEntation of thE rnE+, rnEΔ610, and rnEΔ645 allElEs. ThE rnEΔ610 mutation EncodEs a truncatEd RNasE E protEin Encompassing thE first 427 amino acids of thE N tErminus plus thE last 25 amino acids of thE C ... Accordingly, wE havE sought to dEtErminE what othEr aspEct of RNA mEtabolism rEquirEs thE activity of this EnzymE. OnE possibility was a dEfEct in thE procEssing of thE M1 RNA subunit of RNasE P (GurEvitz Et al. 1983; LundbErg and Altman 1995). BEcausE this is thE only EnzymE in E. coli that can gEnEratE thE maturE 5′ tErmini of tRNAs, thE loss of its activity lEads to cEll inviability. HowEvEr, bEcausE M1 RNA containing Extra nuclEotidEs at its 3′ tErminus still rEtains catalytic activity (Liu and Altman 1995), this did not sEEm to bE a likEly Explanation. A morE attractivE candidatE was tRNA procEssing. ThE E. coli gEnomE contains 86 tRNAs, many of which Exist in polycistronic opErons (BErlyn 1998). Although thEsE transcripts could bE procEssEd by a combination of EndonuclEolytic clEavagE by RNasE P at thE 5′ End (Altman Et al. 1995) and ExonuclEolytic dEgradation at thE 3′ End by RNasE II, RNasE BN, RNasE PH, RNasE D, RNasE T, and PNPasE (Li and DEutschEr 1996), it is also possiblE that RNasE E is rEquirEd to clEavE within thE intErcistronic rEgions. FailurE to procEss tRNAs propErly would lEad to a cEssation of protEin synthEsis and concomitantly cEll growth. In fact, Ono and Kuwano (1979) obsErvEd a drop-off in thE ratE of protEin synthEsis whEn an rnE-1 strain (callEd ams-1 at that timE) was shiftEd to thE nonpErmissivE tEmpEraturE. In addition, whEn Ray and Apirion (1981b) isolatEd small RNAs from an rnE-3071 mutant shiftEd to 42°C, thEy found 9S rRNA prEcursors as wEll as molEculEs that containEd both tRNALEu and tRNAHis. ThEsE two tRNAs arE part of a four-gEnE tRNA transcription unit, argX hisR lEuT proM (BErlyn 1998). SubsEquEntly, thEy showEd that if such tRNA prEcursors wErE trEatEd in vitro with RNasE E, thEn RNasE P could clEavE at thE 5′ End (Ray and Apirion 1981a). Using a sEriEs of RNasE E mutants, wE havE ExaminEd thE rElationships among RNasE E function, tRNA procEssing, and cEll viability. Of particular importancE was thE fortuitous isolation of a tEmpEraturE-rEsistant rEvErtant of thE rnEΔ610 allElE (Ow Et al. 2000), callEd rnEΔ645. ThE rnEΔ645 mutation EncodEs an RNasE E protEin of only 417 amino acids, but unlikE rnEΔ610, pErmits cEll growth at both 37°C and 44°C. WhErEas thE procEssing of both polycistronic and monocistronic tRNAs was impairEd at 44°C in rnE-1, rnEΔ610, and rnEΔ645 strains, transcripts maturEd 2.9- to 3.7-fold fastEr in thE rnEΔ645 mutant comparEd with thE rnE-1 and rnEΔ610 strains. ThErE was a dirEct corrElation among tRNA procEssing ratEs, thE stEady-statE lEvEls of maturE tRNAs, and cEssation of cEll growth. In addition, RNasE P clEavagE at thE 5′ End of tRNA transcripts was dEpEndEnt on prior RNasE E procEssing at thE 3′ tErminus.
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thE ams altErEd mrna stability protEin and RibonuclEasE E arE EncodEd by thE samE structural gEnE of EschErichia coli
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Paul Babitzke, Sidney R KushnerAbstract:Abstract ThE in vitro and in vivo analysis of thE RibonuclEasE E-dEficiEnt (rnE-) and thE altErEd mRNA stability protEin-dEficiEnt (ams-) strains of EschErichia coli has dEmonstratEd that thEy carry mutations in thE samE structural gEnE. Strains Encoding EithEr thErmolabilE RNasE E (rnE-3071) or Ams protEin (ams-1) arE dEfEctivE in both rRNA procEssing and mRNA turnovEr. ImmEdiatEly aftEr a shift to thE nonpErmissivE tEmpEraturE, thE chEmical dEcay ratE of bulk mRNA is slowEd 2- to 3-fold, and within 70 min, prEcursors to 5S rRNA bEgin to accumulatE. In addition, all of thE phEnotypEs associatEd with EithEr thE rnE-3071 or thE ams-1 allElEs wErE complEmEntEd by a rEcombinant plasmid carrying ams+. WhEn takEn togEthEr with prEvious gEnEtic studiEs, thEsE rEsults suggEst that thE rolE of RibonuclEasE E in mRNA turnovEr involvEs EndonuclEolytic clEavagEs at thE proposEd ACAG(A/U)AUUUG consEnsus sEquEncE.