分子生物学英文版教学课件：Ch 14 Translation

1、2022-6-91Molecular Biology of the Gene, 5/E - Watson et al. (2004)Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the GenomePart IV: RegulationPart V: Methods2022-6-92Part III: Expression of the GenomenThis part concerned with one of the greatest challenges

2、in understanding the genehow the gene is expressed.2022-6-93Ch 12: Mechanisms of transcription Ch 13: RNA splicingCh 14: TranslationCh 15: The genetic codeThe revised central dogmaRNA processing基基因因组组的的保保持持基基因因组组的的表表达达学时分配和教学日历学时分配和教学日历课程简介从相互认识到中心法则课程简介从相互认识到中心法则6DNA和和RNA的结构的结构3基因组的保持：基因组的保持：DNA复制复

3、制6基因组的表达：转录基因组的表达：转录 （小考（小考1）6基因组的表达：基因组的表达： RNA剪接与转录后加工剪接与转录后加工6基因组的表达：翻译与遗传密码基因组的表达：翻译与遗传密码（文献报告）（文献报告）6基因调控：原核调控基因调控：原核调控（小考（小考2）6基因调控：真核调控基因调控：真核调控13基因调控：真核调控基因调控：真核调控2RNA干扰与干扰与miRNA调控调控（秀）（秀）3分子生物学技术分子生物学技术6平时小考两次平时小考两次3Molecular Biology Course2022-6-97What is translation? -it is the story abou

4、t decoding the genetic information contained in messenger RNA (mRNA) into proteins. 2022-6-98Questions addressed in this chapternWhat are the main challenges of translation and how do organisms overcome them? nWhat is the organization of nucleotide sequence information in mRNA?nWhat is the structure

5、 of tRNAs, and how do aminoacyl tRNA synthetases recognize and attach the correct amino acids to each tRNA?nHow does the ribosome orchestrate the translation process?2022-6-99Translation extremely costsIn rapid growing bacterial cells, protein synthesis consumesn80% of the cells energyn50% of the ce

6、lls dry weightWhy?2022-6-910The main challenges of translation nThe genetic information in mRNA cannot be recognized by amino acids. nThe genetic code has to be recognized by an adaptor molecule (translator), and this adaptor has to accurately recruit the corresponding amino acid. 2022-6-911Translat

7、ion machinery1.mRNAs (5% of total cellular RNA)2.tRNAs (15%)3.aminoacyl-tRNA synthetases (氨酰tRNA合成酶)4.ribosomes (100 proteins and 3-4 rRNAs-80%)2022-6-912Outline Topics 1-4: Four components of translation machinery. T1-mRNA; T2-tRNA; T3-Attachment of amino acids to tRNA (aminoacyl-tRNA synthetases);

8、 T4-The ribosomeTopic 5-6: Translation process. T5-initiation; T6-elongation; T7-termination. Topic 8: Translation-dependent regulation of mRNA and protein stability2022-6-913Topic 1: mRNAnOnly a portion of each mRNA can be translated.nThe protein-coding region of the mRNA consists of an ordered ser

9、ies of 3-nt-long units called codons that specify the order of amino acids.2022-6-9141-1 polypeptide chains are specified by ORF nThe protein coding region of each mRNA is composed of a contiguous, non-overlapping string of codons called an opening reading frame (ORF) .nEach ORF begins with a start

10、codon and ends with a stop codon.Message RNA2022-6-915The start codon the first codon of an ORFIn bacteria : AUG, GUG, or UUG (5-3)In eukaryotic cells: 5-AUG-3Functions:1.Specifies the first amino acid to be incorporated into the growing polypeptide chain.2.Defines the reading frame for all subseque

11、nt codons. 2022-6-916Prokaryotic mRNA (polycistrionic)Eukaryotic mRNA (monocistrionic)2022-6-917Fig 14-1 Three possible reading frames of the E. coli trp leader sequence2022-6-9181-2 Prokaryotic mRNAs have a ribosome binding site that recruits the translational machineryMessage RNA1-3 Eukaryotic mRN

12、A are modified at their 5 and 3 ends to facilitate translation. 2022-6-919nRibosome binding site (RBS) or SD-sequence in prokaryotic mRNA, complementary with the sequence at the 3 end of 16S rRNA. Fig 14-2-a structure of prokaryotic mRNA2022-6-920 Once Kozak sequencenEukaryotic mRNA uses a methylate

13、d cap to recruit the ribosome. Once bound, the ribosome scans the mRNA in a 5-3 direction to find the AUG start codon.nKozak sequence increases the translation efficiency.nPoly-A in the 3 end promotes the efficient recycling of ribosomes.Fig 14-2-b2022-6-921Fig 1-292022-6-922Topic 2: tRNAAt the hear

14、t of protein synthesis is the translation of nucleotide sequence information into amino acids. This work is accomplished by tRNA.2022-6-9231.The are many types of tRNA molecules in cell (40).2.Each tRNA molecule is attached to a specific amino acids (20) and each recognizes a particular codon, or co

15、dons (61), in the mRNA.3.All tRNAs end with the sequence 5-CCA-3 at the 3 end, where the aminoacyl tRNA synthetase adds the amino acid.2-1: tRNA are adaptors between codons and amino acidsTRANSFER RNA2022-6-9241.tRNAs are 75-95 nt in length. 2.There are 15 invariant and 8 semi-invariant residues. Th

16、e position of invariant and semi-variant nucleosides play a role in either the secondary and tertiary structure. Primary structure2022-6-925Fig 14-3 unusual bases4.Pseudouridine ( U) is a modified base. These modified bases in tRNA lead to improved tRNA function2022-6-926nThe cloverleaf structure is

17、 a common secondary structural representation of tRNA molecules which shows the base paring of various regions to form four stems (arms) and three loops. 2-2: tRNAs share a common secondary structure that resembles a cloverleafTRANSFER RNA2022-6-927Fig 14-4 the secondary structure2022-6-9282-3: tRNA

18、s have an L-shaped 3-D structureFig 14-5 the 3-D structure of tRNAD loop U loop2022-6-929Formation of the 3-D structure : 9 hydrogen bonds (tertiary hydrogen bonds) mainly involving in the base paring between the invariant bases help the formation of tRNA tertiary structure. 2022-6-930The cloverleaf

19、 structure of a tRNA. The tRNA is drawn in the conventional cloverleaf structure, with the different components labeled. 15 invariant nucleotides (A, C, G, T, U, Y, where Y Y =pseudouridine) and 8 semi-invariant nucleotides (abbreviations: R, purine; Y, pyrimidine) are indicated. Optional nucleotide

20、s not present in all tRNAs are shown as smaller dots. The standard numbering system places position 1 at the 5 end andposition 76 at the 3 end; not all of the optional nucleotides are included. The invariant and semi-invariant nucleotides are at positions 8, 11, 14, 15, 18, 19, 21, 24, 32, 33, 37, 4

21、8, 53, 54, 55, 56, 57, 58, 60, 61, 74, 75 and 76. The nucleotides of the anticodon are at positions 34, 35 and 36. /books/2022-6-931nBase pairing between residues in the D-and T-arms fold the tRNA molecule into an L-shape, with the anticodon loop at one end and the amino ac

22、id acceptor site at the other (Fig. 14-5). The base pairing is strengthened by base stacking interactions. 2022-6-932Topic 3: attachment of amino acids to tRNAnAmino acids should be attached to tRNA first before adding to polypeptide chain. ntRNA molecules to which an amino acid is attached are said

23、 to be charged, and tRNAs lacking an amino acid are said to uncharged. 2022-6-9333-1 tRNAs are charged by attachment of an amino acid to the 3 terminal A of the tRNA via a high energy acyl linkagenEnergy: The energy released when the high-energy bond is broken helps drive the peptide bond formation

24、during protein synthesis.nEnzyme: catalyzing the reaction has three binding sites for ATP, amino acid and tRNA. ATTACHMENT OF AMINO ACIDS TO tRNA2022-6-9343-2 Aminoacyl tRNA synthetases charge tRNA in two steps 1. Adenylylation (腺苷酰化) of : transfer of AMP to the COO- end of the amino acids.2. chargi

25、ng: transfer of the adenylylated amino acids to the 3 end of tRNA, generating aminoacyl-tRNAs (charged tRNA).ATTACHMENT OF AMINO ACIDS TO tRNA2022-6-935Class I: attach the amino acids to the 2OH of the tRNA, and is usually monomeric.Class II: attach the amino acids to the 3OH of the tRNA, and is usu

26、ally dimeric or tetrameric.There are two classes of tRNA synthetases.2022-6-936Step 1-Adenylylation of amino acids: the aminoacyl-tRNA synthetase attaches AMP to the-COOH group of the amino acid utilizing ATP to create an aminoacyl (氨酰的) adenylate (腺苷酸) intermediate. . This step is also called activ

27、ation of amino acids p652022-6-9372022-6-9382022-6-939Step 2- tRNA charging: transfer of the adenylated amino acid to the 3 end of the appropriate tRNA via the 2 or 3-OH group, and the AMP is released as a result.2022-6-940Nature structural and Molecular Biology, 2005, 12:915-9222022-6-9413-3： each

28、aminoacyl tRNA synthetase attaches a single amino acids to one or more tRNAsnEach of the 20 amino acids is attached to the appropriate tRNA (s) by aminoacyl-tRNA synthetases.nMost amino acids are specified by more than one codon, and by more than one tRNA as well. ATTACHMENT OF AMINO ACIDS TO tRNA20

29、22-6-942nThe same synthetase is responsible for charging all tRNAs for a particular amino acid (one synthetaseone amino acid).nConsequently, most organisms have 20 synthetases for 20 different amino acids.2022-6-9433-4 tRNA synthetases recognize unique structure features of cognate tRNAs ATTACHMENT

30、OF AMINO ACIDS TO tRNAnThe recognition has to ensure two levels of accuracy: (1) each tRNA synthetase must the correct set of tRNAs for a particular amino acids; (2) each synthetase must all of these (即由一种synthetase所识别的不同tRNAs) 2022-6-944nThe specificity determinants for accurate recognition are clu

31、sters at two distinct sites: the acceptor stem and the anti-codon loop.2022-6-945Fig 14-8Fig 14-72022-6-946Identity elements (specificity determinants ) in various tRNA molecules2022-6-9473-5 Aminoacyl-tRNA formation is very accurate: selection of the correct amino acidThe aminoacyl tRNA synthetases

32、 discriminate different amino acids according to different natures of their side-chain groups.ATTACHMENT OF AMINO ACIDS TO tRNA2022-6-948Fig. 14-92022-6-9493-6 Some aminoacyl tRNA synthetase use an editing pocket to charge tRNAs with high accuracy. ATTACHMENT OF AMINO ACIDS TO tRNA2022-6-950Isoleucy

33、l tRNA synthetase as an example:1.Its editing pocket near the catalytic pocket allows it to proof read the product of the adenylation reaction (step #1).2.AMP-valine and other mis-bound aa can fit into this editing pocket and get hydrolyzed. But AMP-Ile is too big to fit in the pocket. Thus, the bin

34、ding pocket serves as a molecular sieve to exclude AMP-valine etc.ValIle2022-6-951Therefore, Ile-tRNA synthetase discriminates against valine twice: the initial binding and adenylylation of the amino acid, and then the editing of the adenylylated amino acid. Each step discriminates by a factor of 10

35、0, and the overall selectivity is about 10,000-fold.2022-6-9521. Ribosome recognize tRNAs but not amino acids (how to prove?). 2. It is responsible to place the charged tRNAs onto mRNA through base pairing of the codon in mRNA and anticodon in tRNA. 3-7 Ribosomes is unable to discriminate between co

36、rrectly or incorrectly charged tRNAs (是否携带正确的氨基酸)ATTACHMENT OF AMINO ACIDS TO tRNA2022-6-953Topic 4: the ribosome1.Ribosome composition2.Ribosome cycle3.Peptide bond formation4.Ribosome structure2022-6-9544-1 the ribosome is composed of a large and a small subunitnThe large subunit contains the pept

37、idyl transferase center, which is responsible for the formation of peptide bonds.nThe small subunit interacting with mRNA contains the decoding center, in which charged tRNAs read or “decode” the codon units of the mRNA.RIBOSOMES2022-6-955Fig 14-13* Ribosome2022-6-9564-2: the large and the small sub

38、units undergone association and dissociation during each cycle of translation.RIBOSOMES2022-6-957nRibosome cycles: In cells, the small and large ribosome subunits associate with each other and the mRNA, translate it, and then dissociate after each round of translation. This sequence of association a

39、nd dissociation is called the ribosome cycle.2022-6-958Fig 14-14 Overview of the events of translation/ribosome cycle2022-6-959Polysome/polyribosome: an mRNA bearing multiple ribosomes Each mRNA can be translated simultaneously by multiple ribosomes Fig 14-15 A polyribosome2022-6-9602022-6-961RIBOSO

40、MES4-3 New amino acids are attached to the C-terminus of the growing polypeptide chain.Protein is synthesized in a N- to C- terminal direction4-4 Peptide bonds are formed by transfer of the growing peptide chain from peptidyl- tRNA to aminoacyl-tRNA.2022-6-962Fig 14-162022-6-963The structure of the

41、ribosome 4-5 Ribosomal RNAs are both structural and catalytic determinants of the ribosomes 4-6 The ribosome has three binding sites for tRNA.4-7 Channels through the ribosome allow the mRNA and growing polypeptide to enter and/or exit the ribosome.RIBOSOMES4-5: Ribosome structure2022-6-964Fig 14-17

42、 two views of the ribosome2022-6-9654-6 Three binding site for tRNAsFig 14-18A site: to bind the aminoacylated-tRNAP-site: to bind the peptidyl-tRNAE-site: to bind the uncharged tRNA 2022-6-966Fig 14-19 3-D structure of the ribosome including 3 bound tRNA2022-6-9674-7 Channel for mRNA entering and e

43、xiting are located in the small subunit (see Fig. 14-18)There is a pronounced kink in the mRNA between the two codons at P and A sites. This kink places the vacant A site codon for aminoacyl-tRNA interaction. Fig 14-202022-6-9684-7 Channel for polypeptide chain exiting locates in the large subunitTh

44、e size of the channel only allow a very limited folding of the newly synthesized polypeptideFig 14-212022-6-969Translation processT5: Initiation of translationT6: Elongation of translationT7: termination of translation Watch the animation on your study CD2022-6-970Questions1.Compare the mechanism of

45、 translation initiation in prokaryotes and eukaryotes (similarity and difference)2.How do aminoacyl-tRNA synthetases and the ribosomes contribute to the fidelity of translation, respectively? 2022-6-971Overview of the events of translationTermination Elongation InitiationFig 14-142022-6-972T5: Initi

46、ation of translation Initiation in prokaryotic cells (1-3)Initiation in eukaryotic cells (4-6)2022-6-9735-1 Prokaryotic mRNAs are initially recruited to the small subunit by base pairing to rRNA.INITIATION OF TRANSLATIONFig 14-23RBS is also called ShineDalgarno sequence2022-6-9745-2 A specialized tR

47、NA (initiator tRNA) charged with a modified methionine (f-Met) binds directly to the prokaryotic small subunit.INITIATION OF TRANSLATIONFig 14-24 2022-6-9755-3 Three initiator factors direct the assembly of an initiation complex that contains mRNA and the initiator tRNA.INITIATION OF TRANSLATION1. F

48、ormation of the 30S initiation complex: IFs1-3 + 30S + mRNA + fmet-tRNA.2. Formation of the 70S initiation complex: 50S + 30S + mRNA + fmet-tRNA2022-6-9762022-6-977Eukaryotic initiation5-4 Eukaryotic ribosomes are recruited to the 5 cap.5-5 The start codon is found by scanning downstream from the 5

49、end of the mRNA.INITIATION OF TRANSLATIONFigs 14-26 and -272022-6-9785-6 Translation initiation factors hold eukaryotic mRNAs in circlesTry to explain how the mRNA poly-A tail contributes to the translation efficiency?INITIATION OF TRANSLATIONFig 14-292022-6-979T6: Translation elongation 1. Aminoacy

50、l-tRNA binding to A site2. Peptide bond formation3. Translocation2022-6-9806-1 Aminoacyl-tRNAs are delivered to the A site by elongation factor EF-TuELONGATION OF TRANSLATIONFig 14-302022-6-9816-2 The ribosome uses multiple mechanisms to select against incorrect aminoacyl-tRNAsELONGATION OF TRANSLAT

51、IONFig 14-31a2022-6-982Fig 14-31b2022-6-983Fig 14-31c2022-6-9846-3 Ribosome is a ribozyme (重点，重点，催化如何发生？催化如何发生？)ELONGATION OF TRANSLATIONFig 14-32Fig 14-162022-6-985ELONGATION OF TRANSLATION2022-6-9866-4 & 5 Elongation factor EF-G drive translocation of the tRNAs and the mRNA by displacing the t

52、RNA bound to the A site ELONGATION OF TRANSLATION2022-6-987EF-G mimics a tRNA molecule so as to displace the tRNA bound to the A site EF-G-GDPEF-Tu-GDPNP-Phe-tRNAFig 14-352022-6-9886-6 EF-Tu-GDP and EF-G-GDP must exchange GDP for GTP prior to participating in a new round of elongation.ELONGATION OF

53、TRANSLATION2022-6-989Topic 7: Translation termination 2022-6-9907-1 & 2 & 3 The action of Class I and II releasing factors7-1 Class I and Class II releasing factors terminate translation in response to stop codons. TERMINATION OF TRANSLATION2022-6-9917-2 Class I releasing factors (bacterial

54、RF1 and RF2, eukaryotic eRF1) recognize stop codons by its peptide anticodon and trigger the release of the peptidyl chain by the conserved GGQ motif.(Figure 14-37). RF1 has a structure resembles tRNA (Figure 14-38), explaining why it can enters A site. TERMINATION OF TRANSLATION2022-6-9927-3 Class

55、II releasing factor remove Class I releasing factor from the A site. And this function is controlled by GDP/GTP exchange and GTP hydrolysis (Figure 14-39). TERMINATION OF TRANSLATION2022-6-9937-4 Recycling of the ribosome by a combined effort of RRF (ribosome recycling factor), EF-Tu-GTP and IF3. (F

56、igure 14-40)TERMINATION OF TRANSLATION2022-6-994Topic 8: translation dependent regulation of mRNA and protein stability nHere regulation refers cellular processes that deal with defective mRNA and their translated product.2022-6-9958-1: The SsrA RNA rescues (拯救拯救) ribosomes that translate broken mRNAs lacking a stop codon (prokaryotes)1.The ribosomes are trapped or stalled o