生化2014 c17 protein translation.ppt

Biochemistry,Dept. of Biochemistry and Molecular Biology,Professor Wu Yaosheng 2014-06,Chapter 17,Protein Biosynthesis,Protein Biosynthesis,Genes,Gene expression,?,A G C T,A G C U,20种 AA,4,Questions,1.What are requirements for one protein biosythesis in eukaryote cells?,4. Studies have shown that the occurrence of a disease associated with abnormal changes of the structure of a protein in hepatocytes. I ask, how to identify this protein structure has changed? Whether to put forward your ideas?,3.How to synthesize a molecule of hemoglobin with biological functions by gene expression process?,2. How many sequences of DNA could code for the fragment of one peptide Glu-Glu-Met-Trp-Asp？,5,Section 1 Protein Biosynthesis System,Major Topics,Section 2 Linkage of Amino Acids with tRNA,Section 4 Modification and Target Transportation of Post-translation,Section 5 Interference and Inhibition of Protein Biosynthesis,Section 3 Biosynthesis Process of Polypeptide chain,6,Key Points,Functions of mRNA, tRNA, rRNA during Pr synthesis, genetic codon characters Action characters of aminoacyl-tRNA synthetase Comparison of the translation process in prokaryote and in eukaryote Molecular chaperone and modification types post-translation Signal peptides and their actions, directional transportation characteristics Mechanism of translation inhibition by antiboitics, toxins, and interferons,7,Biological Synthesis Process of Protein,The activation and transfer of amino acids,The synthesis of polypeptide,The modification of post-translation,Initiation,Elongation,Termination,Entry of aminoacyl-tRNA,Formation of peptide bond,Translocation of ribosome,Ribosomal cycle,8,Section One,Protein Biosynthesis System,Distinguish monocistron and polycistron; Wobble, Degeneracy of codons,Key points,Functions of mRNA, tRNA, ribosome; ORF, codons and their characters,Difficulties,10,Requirements of Protein Biosynthesis,Various RNAs: mRNA、 rRNA 、tRNA,Substrate： amino acids,Protein factors: Initiation factors,Elongation factors,Termination factors,Enzyme: aminoacyl-tRNA synthetase peptidyltransferase,Energy supply: ATP, GTP,Inorganic ion: Mg2+,11,1. Messenger RNA (mRNA) and Its Codes,Messenger RNA is a polycistron in prokaryotes, but a monocistron in eukaryotes,Proteins,Protein,Function: As a template of protein synthesis,Cistron: encode one polypeptide,12,Concept of codons,The genetic code is the set of rules that specify how the nucleotide sequence of an mRNA is translated into the amino acid sequence of a polypeptide.,Each amino acid in a protein is represented by three consecutive bases in mRNA, and these three-base combination, or triplets, are called codons .,13,The genetic code consists of 64 triplet codons (A, G, C, U) ， 43 = 64,Termination (stop) codons:,Start codon：,UAA, UAG, UGA,AUG (methionine),14,The Genetic Code,UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG,UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG,UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG,UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG,Phenylalanine Leucine Leucine Valine,Isoleucine Methionine,Serine Proline Threonine Alanine,Tyrosine Stop Histidine Glutamine Asparagine Lysine Aspartic acid Glutamic acid,Cysteine Arginine Serine Arginine Glycine,Stop Tryptophan,15,Characteristics of codons on mRNA,(1) Direction （方向性）,(2) Commaless (连续性）,(3) Degeneracy (简并性）,(5) Wobble （摆动性）,(4) Universality （通用性）,16,Reading frame Reading frame is determined by the AUG initiation codon. Every subsequent triplet is read as a codon until reaching a stop codon.,Met Ala Glu Trp Leu Ser Met Ser · · · stop,17,18,Hemoglobin Wayne (3 terminal frameshift mutation),Normal -globin ACG UCU AAA UAC CGU UAA GCU GGA GCC UCG GUA. Thr Ser Lys Tyr Arg stop,mutated region,Wayne -globin .ACG UCA AAU ACC GUU AAG CUG GAG CCU CGG UAG Thr Ser Asn Thr Val Lys Leu Glu Pro Arg stop,19,Degeneracy,Most amino acids have more than one codon, means degenerate. Codons that specify the same amino acid often differ only in the third base.,A mutation only with a base change in DNA may not result in amino acid change in the encoded protein.,CUA CUG ( Leu Leu ) without affecting translation,20,Degeneracy(简并性),Amino acid No. of codon Amino acid No. of codon,21,Universality,The genetic code is the same in most organisms.,Exceptions are found in mitochondrial genome, where some codons specify different amino acids to those normally encoded by nuclear genes.,In some mitochondria, the UGA codon no longer specifies termination of translation but instead encodes tryptophan.,22,23,Preference,Leucine codons(89) of ribosome protein in E.coli Total 1209 codons UUA UUG CUU CUC CUA CUG 1 2 4 3 0 79,23,Wobble,The first base on the anticodon always pairs with the third base on the codon and not strictly obeys the rules of base-pairing.,Codon-anticodon base-pairing is antiparallel.,Codon-anticodon interactions,One tRNA can interact with more than one codon (therefore 50 tRNAs).,The third position in the codon is frequently degenerate.,24,25,U,摆动配对,Wobble pairing of anticodon with codon,26,one tRNAleu can read two of the leucine codons,27,tRNA is the “adaptor” molecule in protein synthesis.,2.Transfer RNA (tRNA),Two functional sites: (1) acceptor stem CCA-OH 3 terminus can carry amino acid on terminal adenosine. (2) anticodon stem and anticodon loop To recognize the codon on mRNA,28,(1) acceptor stem,(2) anticodon stem and anticodon loop,The secondary structure of tRNA,29,DHU arm,Anticodon,Acceptor arm,arm,30,tRNA,31,prokaryotic ribosome,eukaryotic ribosome,3. rRNAs and Ribosomes,32,Ribosomes was organized in three ways,Polysomes several ribosomes bind to and translate a single mRNA simultaneously Free ribosomes Synthesize proteins of cytosol, nucleus, mitochondria Membrane bound ribosomes Synthesize secreted proteins or membrane protein,33,polypeptide chain,amino acids,Large subunit,The functions of ribosome: For the assembly of amino acids to be a polypeptide chain,34,Questions,1. Degenerate codons differ mostly in,A. the identities of their first base. B. the identities of their second base. C. the identities of their third base. D. the wobble position. E. Choices three and four are both correct.,35,35,Questions,2. Ribosomes select the correct tRNAs,A. and then charge them using the appropriate synthetase. B. then bind to the appropriate mRNA. C. solely on the basis of their anticodons. D. with the least abundant anticodons. E. depending on their abundance in the cytosol.,36,Questions,3. Degenerate of genetic codon means that,A.1 AA has more than 2 codons B.2 codons could be combined as 1 codon C.each amino acid always has several codons D.1 codon can represent different amino acid. E.1 amino acid only has 1 codon.,37,Questions,4. The anticodon Which can pair to the codon CGA on mRNA is,A、GCU B、UCG C、CCA D、UCU E、ACU,38,Section Two,Linkage of Amino Acid with tRNA,The activation and transfer of amino acids,Aminoacyl-tRNA synthetase,Two specificities, To catalyze the formation of ester bond between tRNA and amino acid To proofread the wrong ester bond,Two activities, To recognize the amino acid To recognize the tRNA,40,Amino acid + ATP,Aminoacyl-AMP,PPi,Aminoacyl-tRNA synthetase,41,Aminoacyl-AMP + tRNA,Aminoacyl-tRNA + AMP,42,Ala-tRNAAla,Gly-tRNAGly,fMet-tRNAiMet,Met-tRNAiMet,Formylated methionine,Two high energy phosphate bonds needed,43,The formation of fMet-tRNAimet:,Met in eukaryotes is not formylated,Encoded by initiation codon in prokaryotes is formylated (甲酰化）,44,Questions,1. Hydrolytic editing of incorrectly charged tRNAs,A. involves attack of PPi on the aminoacyl-AMP intermediate. B. occurs at a second active site on the synthetase. C. is a waste of ATP, that could be used for other cellular functions. D. is a necessary step for all of the aminoacyl-tRNA synthetases . E. occurs when the incorrect tRNA binds to the synthetase.,45,Questions,2. Most of the identity elements for correct aminoacylation of tRNAs are found,A. in the anticodon stem and the 3' CCA end B. in the T Loop and the D stem and loop. C. in the anticodon and the acceptor stem. D. solely in the anticodon. E. distributed throughout the structure.,46,Section Three,Biosynthesis Process of Polypeptide chain,Biological Synthesis Process of Protein,The activation and transfer of amino acids,The synthesis of polypeptide,The modification of post-translation,Initiation,Elongation,Termination,Entry of aminoacyl-tRNA,Formation of peptide bond,Translocation of ribosome,Ribosomal cycle,48,1. The initiation of polypeptide synthesis,To form an initiation complex,IF1, IF2, IF3 in prokaryotes More than 9 eIF in eukaryotes,Initiation factors ( IF ) :,49,Prokaryotic and Eukaryotic initiation factors,50,The Initiation of Translation in Prokaryotes,Step 1 Dissociation (解聚) of ribosome subunits,Step 2 mRNA enters the small subunit,Step 3 Binding of fMet-tRNAifMet,Step 4 Binding of large subunit,51,IF-3,IF-1,Step 1 Dissociation (解聚) of ribosome subunits,52,IF-3,IF-1,Step 2 mRNA enters the small subunit,53,Molecule interaction between mRNA and the small subunit in prokaryotes.,16s-rRNA of small subunit,mRNA,SD：Shine-Dalgarno: ribosomal binding site, RBS,54,IF-3,IF-1,Step 3 Binding of fMet-tRNAifMet,55,IF-3,IF-1,IF-2,GTP,GDP,Pi,Step 4 Binding of large subunit,56,IF-3,IF-1,IF-2,-GTP,GDP,Pi,57,The Initiation of Translation in Eukaryotes,Step 1 Dissociation (解聚) of ribosome subunits,Step 3 mRNA enters the small subunit,Step 2 Binding of Met-tRNAiMet,Step 4 Binding of large subunit,58,eIF-3,eIF-2B,60S,40S,eIF-6,Step 1 Dissociation (解聚) of ribosome subunits,59,eIF-1,eIF-6,40S,eIF-3,Step 2 Binding of Met-tRNAiMet,60,eIF-6,40S,eIF-3,Step 3 mRNA enters the small subunit,61,40S,Met,5cap,3PolyA,Stop codon,AUG,60S,Step 4 Binding of large subunit,62,2. The elongation of translation,To elongate the polypeptide chain, a serials of cycles called ribosomal cycles are needed,The entry of aminoacyl-tRNA: one GTP needed,The formation of peptide bond: peptidyl-transferase needed,The translocation: one GTP needed,Including:,63,The elongation factors of translation,Prokaryote Functions Eukaryote,EF-Tu,to help aminoacyl-tRNA entry A site,EF-Ts,to bind with GTP,EF-G,EF2,Translocase, help the movement of peptidyl-tRNA from A site to P site,regulatory subunits,EF-1-,EF-1-,64,Elongation Process in Prokaryotes,To protein elongation cartoon movie,65,Elongation process in eukaryotes,It is identical to that in prokaryotes despite of the different elongation factor The polypeptide chain is elongated by addition of amino acid residues one by one on its carboxyl end with the relative movement of the ribosome on mRNA codon by codon, in 5 3 direction.,66,3.The termination of translation,To release the polypeptide chain from the ribosome.,To untie the ribosome from mRNA, tRNA, and to separate the large subunit from small subunit.,The ribosome can be reused for another biosynthesis process of polypeptide.,67,Termination,RF-3：Promote the binding of RF-1 or RF-2 to ribosome; GTPase activity,RF-1：UAA，UAG,RF-2：UAA，UGA,When termination codon is on the A site, RF recognizes it and start the termination process.,Release factor in eukaryote: eRF , eRF recognizes all of the 3 termination,68,Termination of Protein Biosynthesis in Prokaryotes,69,RF,Release factors,Hydrolase activity,70,Termination of translation,Eukaryote, Termination in eukaryotes is catalyzed by a single eukaryotic release factor., All process of translation termination in eukaryote is similar to that in prokaryote in many ways.,71,Termination of translation, Direction of protein synthesis is N terminus to C terminus., Direction of translation is 5 to 3 along the mRNA.,72,Energy consumption in protein synthesis,1. Activation of amino acid：2 ATP,2. elongation： 2 GTP per peptide bond,A. Entrance：1 GTP,For each peptide bond formation, four high-energy bonds are hydrolyzed.,B. Translocation：1 GTP,73,The significance of polysomes,To enhance the velocity of synthesis of polypeptide chain.,Polysomes,During protein synthesis, a single mRNA molecule is bound by multiple ribosomes in interval of about 100 nucleotides, and polypeptides being synthesized simultaneously. This structure of mRNA with multiple ribosomes attached is called polysomes.,Direction,74,Polysomes seen with electron microscope,75,Questions,1. The pathway of a tRNA during polypeptide elongation on the ribosome is,A. A site P site E site. B. P site A site E site. C. A site P site entry site. D. P site entry site exit site. E. None of the choices are correct.,76,Questions,2. On the ribosome, mRNA binds _; the peptidyl transferase reaction occurs _.,A. between the subunits; on the large subunit. B. between the subunits; on the small subunit. C. to the large subunit; on the small subunit. D. to the small subunit; on the large subunit. E. to the small subunit; between the subunits.,77,Questions,3. In prokaryotes, protein synthesis rates are limited by the rate of mRNA synthesis. RNA synthesis occurs at about 50 nucleotides/sec. Therefore, protein synthesis occurs at about _ amino acids/sec.,A. 10 B. 17 C. 25 D. 34 E. 50,78,Section Four,Post-translation Processing and Targeting,Post-translation Processing,(2) Modification of protein primary structure,(1) Folding of newly synthesis polypeptide,(3) The polymerization of subunits,(4) Target transportation of proteins,80,(1) Folding of newly synthesis polypeptide,A. Molecular chaperones,B. Isomerases,heat-shock protein (HSP ) family,Chaperone, chaperonin,Protein disulfide isomerase (PDI),Peptidyl prolyl cis-trans isomerase (PPI),81,(2) Modification of protein primary structure, hydrolytic modification, Acylation, hydroxylation, methylation or phosphorylation of polypeptide., Removing of Met residues or a few of amino acid residues on the N-end terminus of proteins.,Glycosylation of proteins,Modification of protein on side chain structures,N-end modification,82,N,C,signal peptide,KR,KR,POMC(鸦片促黑皮质素原), pro-opiomelanocortin, is the precursor of several bioactive peptides.,KR is the abbreviation of lysine, arginine,MSH, melanocyte-stimulating hormone(促黑激素）,ACTH, adrenocorticotropic hormone （促肾上腺皮质激素）,-LT, -lipotropin （脂酸释放激素）,83,(3) The polymerization of subunits, Some proteins have quarternary structure but are expressed from different genes.,The typical example is hemoglobin, Therefore, the polypeptide chains would be polymerized to form the quarternary structure after the biosynthesis of polypeptide chain.,84,Post-translational processing,Polymerization of subunits,Oligomer,85,(4) Target transportation of proteins,The final location of a protein results from a process called protein targeting.,The targeting of protein depends on the signal that a polypeptide contains.,Various proteins have diffe