《分子生物学动画》PPT课件.ppt

科恩伯格现年59岁，目前供职 于美国斯坦福大学医学院，他 的父亲阿瑟科恩伯格是1959年 的诺贝尔医学或生理学奖得主 之一。科恩伯格揭示了真核生 物体内的细胞如何利用基因内 存储的信息生产蛋白质，而理 解这一点具有医学上的“基础性” 作用，因为人类的多种疾病如 癌症、心脏病等都与这一过程 发生紊乱有关。 2006年化学诺贝尔奖 罗杰科恩伯格 H1 The genetic code H2 Protein synthesis(translation) in prokaryotes H3 Translation in eukaryotes H4 Protein targeting H5 Protein glycosylation H protein synthesis The genetic code is a triplet code The genetic code is degenerate Universality of the genetic code Reading frames Open reading frames H1 The genetic code 1. Triplet code The relationship between the nucleotide sequence of the mRNA and the amino acid sequence of the polypeptide is called the genetic code. Triplet code The three nucleotides in group called codon 43=64 codons Initiation codon (AUG) stop codon ( UAA, UAG,UGA) New codon: UGA Se-cysteine Triplet code 2.The genetic code is degenerate There are 64 codons, but only 20 amino acids. Degenerate: A single amino acid is coded for by several different codons. Synonyms: Different codons that specify the same amino acid. Degeneracy of the genetic code Codon and anticodon Anticodon: a triplet of bases in a specific tRNA molecule. Each base in the codon base pairs with its complementary base in the anticodon. Wobble base-pairing 3.Universality of the genetic code All living organism used almost the same code. But there are a few differences. E.g. in mitochondrial mRNAs, some codons have different meanings. Different code 4.Reading frames Start: UAG Stop: UAA, UAG, UGA Unit: Triplet Character: Continuous mutant 5.Open reading frames An open reading frame (ORE) is a run of codons that starts with ATG and ends with a termination codon, TGA TAA or TAG. To identify potential coding regions. Coding regions of genes contain relatively long ORFs unlike noncoding DNA where ORFs are comparatively short. Overview Synthesis of aminoacyl-tRNA Initiation of protein synthesis Elongaion Termination H2 translation in prokaryotes 1. Overview Direction: mRNA: 53, Protein: NC Site: ribosome Recognizing: Codon (mRNA) base pairs anticodon (tRNA) via hydrogen bonding Translation phase: initiation, elongation and termination. 2. Synthesis of aminoacyl-tRNA (amino acid activation) Each tRNA molecule has a cloverleaf secondary structure consisting of three stem loops, one of which bears the anticodon at its end. The amino acid is covalently bound to the 3 OH group at the 3 end by aminoacyl synthetase to form aminoacyl-tRNA. The reaction is called amino acid activation. tRNAGly Gly-tRNAGly Question There are 61codons, 20 amino acids. How many are there tRNA and aminoacyl-tRNA synthetase? Synthesis of aminoacyl-tRNA is crucially important for two reasons First : each amino acid must be covalently linked to a tRNA molecule in order to take part in protein synthesis,which depend upon the adaptor function of tRNA. Second: the covalent bond is a high energy bond that enables the amino acid to react with the end of the growing polypeptide chain. The synthesis reaction occurs in two steps The first step is the reaction of amino acid and ATP to form an aminoacyl-AMP. The second step is the aminoacyl group of aminoacyl-AMP is transferred to the 3end of the tRNA molecule to form aminoacyl- tRNA. step one Step two: Aminoacyl-AMP+tRNA-aminoacyl-tRNA +AMP 3.Initiation of protein synthesis Each ribosome has three binding sites for tRNAs; an A site where the incoming aminoacyl-tRNA binds, a P site where the tRNA linked to the growing polypeptide chain is bound, and an E site which binds tRNA prior to its release from the ribosome . Translation in prokaryotes begins by the formation of a 30S initiation complex between the 30S ribosomal subunit, mRNA, initiation factors and fMet tRNA fmet . The 30S subunit binds to the Shine-Dalgarno sequence which lies 5 to the AUG Start codon and is complementary to the 16S rRNA of the small ribosomal subunit. Initiation The ribosome then moves in a 3 direction along the mRNA until it encounters the AUG codon. The 50S ribosomal subunit now binds to the 30S initiation complex to form the 70S initiation complex. In this complex, the anticodon of the fMet-tRNA fMet is base-paired to the AUG initiation codon (start codon) in the P site. Initiation fMet-tRNAfMet 4. Elongation Elongation of the polypeptide chain occurs in three steps: 1.Aminoacyl-tRNA binding 2.Peptide bond formation 3.Translocation Elongation Factor Tu(EF-Tu) 5.Termination Termination codons: UAA, UAG, UGA Release factors: RF1, RF2, RF3 RF1 UAA,UAG RF2 UGA RF3 RF3 Protein synthesis in E.coli Initiation Elongation Termination H3 Translation in eukaryotes Comparison of protein synthesis in prokaryotes and eukaryotes prokaryoticEukaryoticcharacter Polycistron SD sequence AGGAGGU fMet-tRNAfMet EF-Tu, EF- Ts, EF-G RF1, RF2, RF3 Monocistron Kozak sequence ACCAUGG Met-tRNAiMet eEF1,eEF1, eEF2 eRF mRNA Near initiation AUG Initiative tRNA Elongation factors Termination factors 2006年医学和生理学诺贝尔奖 安德鲁法尔(右图)和克雷格梅洛(左图) 安德鲁法尔1959年出生，目前任职于美国 麻省理工学院；克雷格梅洛1960年出生， 目前在美国哈佛大学工作。法尔和梅洛获 奖是因为他们“发现了控制遗传信息流动的 基本机制”。公报指出，RNA干扰已被广泛 用作研究基因功能的一种手段，并有望在 未来帮助科学家开发出治疗疾病的新疗法 。 Overview Secretory protein Plasma membrane proteins Proteins of the endoplasmic reticulum Lysosomal proteins Mitochondrial and chloroplast proteins Nuclear proteins H4 Protein targeting 1.Overview Both in prokaryotes and eukaryotes, newly synthesized proteins must be delivered to specific subcellular location or exported from the cell for correct for activity. This phenomenon is called protein targeting. 2.Secretory proteins Secretory proteins have an N- terminal signal peptide which targets the protein to be synthesized on the rough endoplasmic reticulum. During synthesis it is translocated through the RER membrane into the lumen. Vesicles then bud off from the RER and carry the protein to the Golgi complex, where it becomes glycosylated. Others vesicles then carry it to the plasma membrane. Fusion of these transport vesicles with the plasma membrane then releases the protein to the cell exterior. Secretory proteins 3.Plasma membrane proteins The orientation of the protein in the membrane is determined by topogenic sequences within the polypeptide chain .Type proteins have a cleaved N-terminal signal sequence and a hydrophobic stop-transfer sequence ， Type have an uncleaved N-terminal signal sequence that doubles as the membrane-anchoring sequence, and Type have multiple signal sequences and stop-transfer sequences . 4.Proteins of the endoplasmic reticulum Proteins destined for the ER have an N-terminal signal peptide, are synthesized on the RER, are translocated into the RER lumen and transport- ed by vesicles to the Golgi. Once there, a C- terminal amino acid sequence （ KDEL ） is recognized by a Golgi receptor protein that causes other vesicles to return the protein to the ER. 5.Lysosomal proteins Lysosomal proteins are targeted to the lysosomes via the addition of a mannose 6-phosphate signal that is added in the cis-compartment of the Golgi and is recognized by a receptor protein in the trans-compartment of the Golgi. 6. Mitochondrial and chloroplast proteins Most mitochondria and chloroplast protein are made on free cytosolic ribosomes, released into the mitochondrial matrix requires a matrix-targeting sequence and occurs at sites where the outer and inner mitochondrial membranes come into contact. 7.Nuclear protein Proteins destined for import into the nucleus typically require a nuclear localization signal, four to eight amino acids long (Lys, Arg, Pro), located internally in the protein. Uptake occurs via nuclear pores and requires ATP hydrolysis. 不同蛋白转运特征 名 称特 征 分泌蛋白 膜蛋白 内质网 溶酶体 线粒体、叶 绿体 核蛋白 Three types of protein glycosylation Synthesis of O-linked oligosaccharides Synthesis of N-linked oligosaccharides H5 protein glycosylation 1.Three types of protein glycosylation Many protein synthesized by ribosomes of the RER contain short chains of carbohydrates (oligosaccharides )and are called glycoproteins. The oligosaccharides a