生物化学biochem课件ch15-protein metabolism

1、Protein MetabolismProtein synthesis- Translation The process in which the genetic information present in an mRNA molecule specifies the sequence of amino acids during protein synthesis.以mRNA为直接模板，tRNA为氨基酸运载体,核蛋白体为装配场所，共同协调完成蛋白质生物合成的过程。也就是把mRNA的碱基排列顺序转译成多肽链中氨基酸的排列顺序。Three major advances set the stage

2、 for our present knowledge of protein biosynthesis The site of protein synthesis: ribosomes Amino acid are activated as aminoacyl-tRNA The genetic codeContents1. The genetic code2. Protein synthesis3. Protein targeting and degradation1.The genetic codeThe genetic code was cracked using artificial mR

3、NA templates A codon is a triplet of nucleotides that codes for a specific amino acid. No punctuation between codons Initiation codon: AUG Termination codons (stop codons, nonsense codons): UAA, UAG, UGA A reading frame is a contiguous (邻近的) and nonoverlapping set of three-nucleotide codons in DNA o

4、r RNA. There are 3 possible reading frames in a strand. A reading frame that contains a start codon and a stop codon is called an open reading frame. Reading frame and Open reading frameFeatures of genetic code commaless (连续性) nonoverlapping (读码不重叠) Universal (通用性) Degenerate (简并性) Wobble (摆动性)Codon

5、 and anticodonwobbleWobble allows some tRNA to recognize more than one codon Significance?Balance the requirements for accuracy and speed.密码子的特性密码子的特性 无标点符号、读码不重叠无标点符号、读码不重叠甲硫氨酸天冬酰胺精氨酸谷氨酸甘氨酸AUG AAUAGAG AAGGCCG碱基丢失碱基丢失赖氨酸丙氨酸酪酪氨氨酸酸UU一个碱基突变一个碱基突变但如果是密码子但如果是密码子第三个碱基改变，第三个碱基改变，氨基酸有可能不氨基酸有可能不变。简并性、摆变。简并性、

6、摆动性动性、一定的防突变功能、一定的防突变功能CTranslational frameshifting and RNA editingSome mRNAs are edited before translationRNA editing of the transcript of the cytochrome oxidase subunit II gene from mitochondria of Trypanosoma bruceiRNA editing of the transcript of the gene for the apo-lipoprotein B-100 component

7、of low-density lipoproteinOverlapping gene in different reading frames are found in some viral DNAsGenes within genes. the circular DNA of X174Note: nonoverlapping in codonsPortion of the nucleotide sequence of the mRNA transcript of gene C, A, and K of X1742. Protein synthesisProtein synthesis take

8、s place in the ribosome The ribosome is a complex supermolecular machineBacterial ribosomes contains about 65% rRNA and about 35%proteinStructure of the bacterial ribosome. green rope: mRNA; shades of green: tRNA The rRNAs appear to serve as a framework to which ribosomal proteins are bound. Protein

9、s in the bacterial ribosome serve as either enzymes or structural components in protein synthesis, although the detailed functions of most of these proteins are yet to be elucidated.tRNAs serve as adaptors in translating the language of nucleic acids into the language of proteins Transfer RNA have c

10、haracteristic structural features At least 32 tRNA are required to recognize all the amino acid codons, but some cells use more than 32General cloverleaf (三叶草形) secondary structure of all tRNA. D: dihydrouridine; pseudouridineNucleotide sequence of yeast tRNAAlaThree-dimensional structure of tRNA (倒

11、L型)mRNA serves as an template for translationProcess of protein synthesisStage 1: Activation of amino acidsSynthesis of a polypeptide with a defined sequence demands that two fundamental chemical requirements be met:The carboxyl group of each amino acid must be activated.A link must be established b

12、etween each new amino acid and the information that encodes it in the mRNA.Process of protein synthesis Each enzyme is specific for one amino acid and one or more corresponding tRNA. Mg 2+Amino acid + tRNA + ATPAminoacyl-tRNA + AMP + PPiAminoacyl-tRNA synthetases (氨酰-tRNA合成酶) attach the correct amin

13、o acids to their tRNAGeneral structure of aminoacyl-tRNAProofreading by aminoacyl-tRNA synthetases The aminoacylation of tRNA plishes (1) activation of an amino acid and (2) attachment of the amino acid to an adaptor tRNA that ensures appropriate placement of the amino acid in a growing polypeptide.

14、 The identity of the amino acid attached to a tRNA is not checked on the ribosome, so attachment of the correct amino acid to the tRNA is essential to the fidelity of protein synthesis.A “second genetic code” Interaction between aminoacyl-tRNA synthetases and tRNAs has been referred to as the “secon

15、d genetic code”.Position in blue are the same in all tRNA, and therefore can not be used to discriminate one from another. Other position are known recognition points for one(orange) or more (green) aminoacyl-tRNA synthetasesKnown position in tRNA recognized by aminoacyl-tRNA synthetases.Stage 2: In

16、itiationSpecific amino acid initiates protein synthesisQuestion 1How can Methionine distinguish initiation codon AUG from internal codon AUG?Process of protein synthesisThere are two tRNA for Methionine tRNAMet: for interior positions In bacteria, tRNAfMet: for initiating AUG In eukarotic cells, all

17、 polypeptides synthesized by cytosolic ribosomes begin with a Met residue (rather than fMet), but a specialized initiating tRNA is used that is distinct from the tRNAMet used at AUG codons at interior positions in the mRNA. Polypeptides synthesized by the ribosomes in the mitochondra and chloroplast

18、s, however, begin with a fMet First, methionine is attached to tRNAfMet and then a transformylase transfers a formyl group to the amino group of the Met. Question 2How can the single AUG codon identify both the starting N-formylmethionine(or methionine, in eukaryotes) and Met residues that occur in

19、interior positions in polypeptides? The initiating AUG is guided to its correct position by the Shine-Dalgarno (SD) sequence in the mRNA.SD sequence: four to nine purine residues, eight to 13 bp to the 5 side of the initiation codonFormation of the initiation complex Bacterial ribosomes have three s

20、ites that bind aminoacyl-tRNAs, the aminoacyl or A site, the peptidyl or P site and the exit or E site. The fMet-tRNAfMet is the only aminoacyl-tRNA that binds first to the P site.Initiation in eukaryotic cellsProtein complexes in the formation of a eukaryotic initiation complexStage 3: elongationPe

21、ptide bonds are formed in the elongation stageBinding of an ing aminoacyl-tRNA.Peptide bond formation.Translocation.Process of protein synthesisFirst step in elongation (bacteria): binding of the second aminoacyl-tRNAFormation of first peptideTranslocationProofreading on the ribosome Check if the pr

22、oper codon-anticodon pairing has taken place during the first step of elongation Stage 4: termination and release Termination of polypeptide synthesis requires a special signalProcess of protein synthesisTermination of protein synthesis in bacteriaProcess of protein synthesisRapid translation of a s

23、ingle message by polysomes (polyribosomes多核糖体) In bacteria, transcription and translation are tightly coupled In eukaryotic cells, newly transcribed mRMAs must be transferred out of the nucleus before they can be translated.Electron micrograph and explanatory diagram of a polysomeFour polysomes are

24、shown translating a eukaryotic mRNA molecule simultaneouslyCoupling of transcription and translation in bacteriaStage 5: folding and posttranslational processing The new synthesized polypeptide must fold into its proper three-dimensional conforma-tion. Before or after folding ,the new polypeptide ma

25、y undergo enzymatic processing.Process of protein synthesisPosttranslational processing (modification) Amino-terminal and carboxy-terminal modification, for example, removal of one or more amino acids from the amino terminus. Loss of signal sequences. Proteolytic cleavage. Attachment of oligosacchar

26、ides (寡糖). Modification of individual amino acids.addition of acetyl, phosphoryl, methyl, carboxyl, or other groups to certain amino acid residues. Addition of prosthetic groups (辅基), such as the heme group of cytochrome c. Formation of disulfide cross-links.Posttranslational processing(continued)vP

27、rotein synthesis is inhibited by many antibiotics and toxins Tetracyclines（四环素）: blocking the A site Chloramphenicol（氯霉素）: blocking peptidyl transfer Streptomycin（链霉素）: causing misreading of the genetic code at low concentration, inhibiting initiation at higher concentration.3. Protein targeting and

28、 degradation (转运与降解)Cellular destination of proteinsLysosomal, membrane, or secreted proteinsProteins destined for mitochondra, chloroplast, or the nucleusCytosolic proteinsPosttranslational modification of many eukaryotic proteins begins in the endoplasmic reticulum Most lysosomal, membrane, or sec

29、reted proteins have an amino terminal signal sequence (13 to 36 amino acid residues).Translocation into the ER directed by amino-terminal signal sequences of some eukaryotic proteinsCommon features of signal peptides About 10-15 hydrophobic amino acid residues One or more positively charged residues

30、, usually near the amino terminus preceding the hydrophobic sequence. A short sequence at the carboxyl terminus (near the cleavage site) that is relatively polar, typically having amino acid residues with short side chains (especially Ala) at the position closest to the cleavage site.Directing eukar

31、yotic proteins with the appropriate signals to the ER. SRP: signal recognition particleGlycosylation plays a key role in protein targeting Many proteins are glycosylated in ER. Glycoproteins are often linked to their oligosaccharides (寡糖) through Asn residues (N-linked oligosaccharides). In the Golg

32、i complex, O-linked oligosaccharides are added and N-linked oligosaccharides are further modified.Synthesis of the core oligosaccharide of glycoproteins磷酸多（长）萜醇Pathway taken by proteins destined for lysosomes, the plasma membrane, or secretion Sorting must be based on the basis of structural feature

33、s other than the signal sequence, for example, N-linked oligosaccharides (including their modification ,such as phosphorylated monnose) plays a key role in targeting hydrolases to lysosomes.Proteins are targeting to mitochondra and chloroplasts by similar pathway Precursor proteins destined for mito

34、chondra or chloroplasts have amino-terminal signal sequences that are bound by cytosolic chaperone proteins. The precursor are delivered to receptors on exterior surface of the target organelle, then to a protein channel that usually spans the inner and outer membranes of the organelle. Inside the o

35、rganelle, the signal sequence of the precursor is removed, and the mature protein is refolded.Targeting to the mitochondrial matrixSignal sequences for nuclear transport are not cleavedMovement of macromolecules through nuclear poresRNAs are exported to the cytoplasmRibosomal proteins are imported t

36、o the nucleus and assembled 60S and 40S ribosomal subunits are then exported back to the cytosol.A variety of nuclear proteins imported into the nucleus. The signal sequences that targets a protein to the nucleus (nuclear localization sequence, NLS) are not removed. An NLS may be located almost anywhere along the pri