Probing the Informational and Regulatory Plasticity of a Transcription Factor DNA–Binding Domain 英文参考文献.docVIP

ProbingtheInformationalandRegulatoryPlasticityofa TranscriptionFactorDNA–BindingDomain RyanK.Shultzaberger1¤,SebastianJ.Maerkl2,JackF.Kirsch1,MichaelB.Eisen1,3,4* 1Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, United States of America, 2School of Engineering, Institute of Bioengineering,EcolePolytechniqueFederaledeLausanne(EPFL),Lausanne,Switzerland,3DepartmentofGenomeSciences,GenomicDivision,ErnestOrlandoLawrence BerkeleyNational Lab, Berkeley, California, United States of America, 4Howard Hughes Medical Institute,University of California Berkeley, Berkeley, California, United StatesofAmerica Abstract Transcription factors have two functional constraints on their evolution: (1) their binding sites must have enough information to be distinguishable from all other sequences in the genome, and (2) they must bind these sites with an affinitythatappropriatelymodulatestherateoftranscription.Sincebotharedeterminedbythebiophysicalpropertiesof theDNA–bindingdomain,selectionononewillultimatelyaffecttheother.Wewereinterestedinunderstandinghowplastic theinformationalandregulatoryproperties ofatranscriptionfactor areandhowtranscriptionfactors evolvetobalance theseconstraints.Tostudythis,wedevelopedaninvivoselectionsysteminEscherichiacolitoidentifyvariantsofthehelix- turn-helix transcription factor MarA that bind different sets of binding sites with varying degrees of degeneracy. Unlike previous in vitro methods used to identify novel DNA binders and to probe the plasticity of the binding domain, our selectionsweredonewithinthecontextoftheinitiationcomplex,selectingforbothspecificbindingwithinthegenome andforaphysiologicallysignificantstrengthofinteractiontomaintainfunctionofthefactor.UsingMITOMI,quantitative PCR, and a binding site fitness assay, we characterized the binding, function, and fitness of some of these variants. We observed that a large range of binding preferences, information contents, and activities coul