翻译（第二讲）2

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,免疫学信息网,http:/www.,immuneweb,.com,New Interaction Between Old Partners,fMet,-,tRNA,eIF1A,eIF5B,Marintchev,et al.,2003,1,Delivery of,fMet,-,tRNA,via eIF2,eIF2/GTP delivers,fMet,-,tRNA,to the 40S particle and places it into the P-site,eIF2 is a,trimeric,complex(,a,b,g,)of 127,kDa,Binds,fMet,-,tRNA,when GTP is bound,eIF2B is the,Guanylate,exchange factor(GEF)for eIF2,eIF2B is a,pentamer,of 261,kDa,no structure is known,Ternary complex formation is inhibited by,phosphorylation,of eIF2,a,at Ser 51,The ternary complex binds the 40S subunit to form the 40S,preinitiation,complex(43S initiation complex),eIF3 is needed for binding the ternary complex,Ternary complex stays on the ribosome until AUG,codon,is reached(after scanning).At this point,hydrolysis of eIF2-bound GTP is catalyzed by eIF5,eIF2-GDP leaves and,fMet,-,tRNA,stays on the ribosome,2,More on eIF2,eIF2 is a 127,kDa trimeric,complex highly conserved in,eukaryotes,eIF2,a,(36,kDa,)has a crucial,phosphorylation,site at S51,eIF2,(39,kDa,)function not clear,eIF2,(52,kDa,)GTP-binding,There is no structure yet of entire eIF2,N-terminus of eIF2,a,has OB fold,crystal structure of N-terminal half has been solved(Jon,Clardy,lab,JBC 277,17057(2002),Ser51 sits on a flexible loop,Ser 51,phosphorylation,leads to a conformational change that causes formation of a tight complex with eIF2B and initiation is inhibited,3,eIF2 consists of three subunits,a,-subunit,P-Ser,51,315,b,-subunit,Lys,blocks,Zn finger motif,14,80,124,281,305,333,g,-,subunit,GTP binding motif,48,134,190,472,eIF5,tRNA,i,Met,eIF2B,eIF2B,PKR,HRI,GCN2,PERK,4,Significance of Ser 51,phosphorylation,Ser51 is,phosphorylated,by,kinases,such as,double-stranded RNA-,activted,protein,kinase,PKR,PERK=PKR-like ER,kinase,Heme,-,regulatd,eIF2,a,kinase,(HRI=,heme,-regulated,inhibvitor,),GCN2 in yeast,Reversible,phosphorylation,provides the cells with a mechanism to regulate the the amount of,mRNA,rapidly and reversibly in response to a variety of stimuli,PKR:response to double-stranded RNA virus,PKR-induced eIF2,a,phosphorylation,can cause apoptosis,Iron deficiency-,heme,deficiency activates HRI-Ser51,phosphorylation,and,downregulation,of translation in erythrocytes,PERK regulates translation in the ER in response to stress,5,eIF4A,m7Gppp,AAAAAA,AUG,eIF4E,eIF4G,Recruitment of,mRNA,to the 43S ribosomal subunit,eIF3,40,S,eIF2,Met-,tRNA,i,eIF1A,eIF4F=eIG4G/eIF4E/eIF4A,eIF4G is a scaffold protein that binds eIF4E,eIF4A,Pabp,Mnk,-,kinase,eIF3,eIF4E is cap-binding protein,eIF4A is RNA,helicase,6,cap,4,EBP,eIF4G,Structure of eIF4E,Matsuo et al.1997,N,35,7,Residues 1-33 are unstructured in free eIF4E,What happens when eIF4G binds?,8,eIF4G1,393-490,folds upon binding eIF4E,free,bound to eIF4E,Hershey,McWirther,Gross,Wagner,Alber,Sachs,JBC 1999,9,eIF4E/eIF4G/cap complex,10,N,C,C,N,cap,eIF4E,eIF4G,Summary,eIF4E/eIF4G/cap complex,N-terminal segment of eIF4E folds upon eIF4G binding,eIF4G wraps around N-terminus of eIF4E,Binding induces mutual folding to form a 35,kDa,complex,11,m7Gppp,AAAAAA,AUG,eIF4E,eIF4G,eIF3,40,S,Recruitment of,mRNA,to the 43S ribosomal subunit,eIF4A,Formation of this complex is highly regulated,Gross et al.2003,Marcotrigiano,et al.2001,Middle domain is a,HEAT domain,Burley et al.2003,NTD-eIF4A,CTD-eIF4A,eIF4A is a DEAD-box protein,Marcotrigiano,et al.1997,Matsuo e al.1997,12,Regulation of translation initiation by 4E-BP,eIF4E binding to eIF4G is inhibited by 4E-BP,eIF4G and 4E-BP share a consensus binding sequence,YxxxxL,F,Growth factors or hormones may cause,hyperphosphorylation,of 4EBP,dissociation and formation of,preinitiation,complex,Translation initiated,13,4,EBP is unstructured and exhibits only minor changes upon binding eIF4E,free,complex,14,YXXXXL,F,15,16,Hierarchical,phosphorylation,of 4E-BP,17,Initiation via Internal Ribosome Entry Sites,Some,mRNAs,have 5UTRs that act as direct binding sites for small ribosomal particle-in concert with the middle domain of eIF4G,First identified for viral,RNAs,Hepatitis C virus(HCV),Classic swine fever virus(CSFV),Foot and mouth disease virus,Now several cellular proteins have been found to be translated via,IRESs,eIF4G,FGF2,VEGF etc.,18,Preinitiation,complex in translation initiation,eIF4G,Recruitment of 40S to,mRNA,via,eIF3eIF4GeIF4Em7G,mRNA,Recruitment of 40S to,mRNA,via eIF3eIF4G,Pabp,poly(A),mRNA,Recruitment of 40S to,mRNA,via eIF3eIF4GIRES,4,B/4H,Alan Sachs et al.,19,Eukaryotic,Translation Initiation-Summary,Complex,eukaryotic,translation initiation is to provide more control over protein production,Many forms of cancer have elevated levels of initiation factors,20,Eukaryotic,Translation Initiation-details,Dissociation of ribosome binding eIF1A and eIF3 to 40S,Recruit ternary complex(,fMet,-,tRNAi,eIF2,GTP)to 40S,Recruit,mRNA,to 43S-46S particle,46 S scans,mRNA,to reach AUG,Initiation factors leave and 80S joins,Elongation begins,Highly control