Structural Basis Of Class ? CAP-dependent Bacterial Transcription Activation

Transcription activation is the basis of gene expression,gene regulation,and development.The process of transcription activation requires the participation of RNA polymerase,transcription activator protein,DNA and ribonucleotides.Catabolite activator protein(CAP)in E.coli(Escherichia coli)is the first purified transcription activator protein and the first transcription activator protein,of which a threedimensional structure was determined.In E.coli,it can activate the transcription of more than 100 genes and is a classic transcription activator protein.CAP is also known as c AMP receptor protein(CRP).After the binding of allosteric factor c AMP,the conformation of CAP changes to activate transcription.The simplest promoters dependent on CAP to activate transcription only require CAP,and no other auxiliary factor is required to activate transcription.There are two classes of CAP-dependent promoters based on their different CAP binding sites: at class I CAP-dependent promoters,such as the lac promoter and the artificial promoter CC(-61.5),the DNA binding site of CAP is located upstream of the DNA binding site of RNA polymerase;at class II CAP-dependent promoters,such as the gal promoter and the artificial promoter CC(-41.5),the DNA binding site of CAP partially overlaps the DNA binding site of RNA polymerase.The structure of a complete transcription activation complex(TAC)is of great significance for exploring the mechanism of transcription activation.Previous work has reported the structures of E.coli class I CAP-dependent TAC and Thermus thermophilus class II CAP-dependent TAC.In contrast to E.coli CAP,Thermus thermophilus CAP does not require c AMP for its function.Since E.coli is a model organism and most relevant genetic studies were done in E.coli,determining the structure of E.coli class II CAP-dependent TAC would provide information for the mechanism study of transcription activation.In order to obtain an E.coli class II CAP-dependent TAC,we designed a nucleic acid scaffold that can represent a complete E.coli class II CAP-dependent promoter at positions ranging from-63 to +15.This scaffold contains a conserved CAP binding site,a conserved extended-10 element,a conserved-10 element,a conserved discriminator element,a conserved core recognition element,and a 13 bp transcription bubble.To obtain the structure similar to that in physiological condition,E.coli class II CAPdependent TAC was reconstituted with scaffold,purified RNA polymerase,CAP,and c AMP,frozen in liquid nitrogen,observed with cryo-electron microscopy,and the data were processed in RELION.In the end,we resolved the structure of E.coli class II CAP-dependent TAC with a resolution of 4.0 ?.This structure reveals the interaction between RNA polymerase and CAP,the interaction between CAP and DNA,and the interaction between RNA polymerase and DNA during transcription activation.Our structure provides a structural basis for understanding class II CAP-dependent transcription activation.In addition to the simplest class I and class II CAP-dependent transcription activation,other transcription activator proteins require co-factors,which may have some extra interactions with RNA polymerase,so the structures of more complicate transcription activation complexes still deserve further investigation.