1、2022/8/311Molecular 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: Methods2022/8/312Part III: Expression of the GenomeThis part concerned with one of the greatest challenges

2、 in understanding the genehow the gene is expressed.2022/8/313Ch 12: Mechanisms of transcription Ch 13: RNA splicingCh 14: TranslationCh 15: The genetic codeThe revised central dogmaRNA processing基因組的保持基因組的表達學(xué)時分配和教學(xué)日歷內(nèi)容學(xué)時課程簡介從相互認識到中心法則6DNA和RNA的結(jié)構(gòu)3基因組的保持：DNA復(fù)制6基因組的表達：轉(zhuǎn)錄 （小考1）6基因組的表達： RNA剪接與轉(zhuǎn)錄后加工6基因組的

3、表達：翻譯與遺傳密碼（文獻報告）6基因調(diào)控：原核調(diào)控（小考2）6基因調(diào)控：真核調(diào)控13基因調(diào)控：真核調(diào)控2RNA干擾與miRNA調(diào)控（秀）3分子生物學(xué)技術(shù)6平時小考兩次3CHAPTER 14TranslationMolecular Biology Course2022/8/317What is translation? -it is the story about decoding the genetic information contained in messenger RNA (mRNA) into proteins. 2022/8/318Questions addressed in t

4、his chapterWhat are the main challenges of translation and how do organisms overcome them? What is the organization of nucleotide sequence information in mRNA?What is the structure of tRNAs, and how do aminoacyl tRNA synthetases recognize and attach the correct amino acids to each tRNA?How does the

5、ribosome orchestrate the translation process?2022/8/319Translation extremely costsIn rapid growing bacterial cells, protein synthesis consumes80% of the cells energy50% of the cells dry weightWhy?2022/8/3110The main challenges of translation The genetic information in mRNA cannot be recognized by am

6、ino acids. The genetic code has to be recognized by an adaptor molecule (translator), and this adaptor has to accurately recruit the corresponding amino acid. 2022/8/3111Translation machinerymRNAs (5% of total cellular RNA)tRNAs (15%)aminoacyl-tRNA synthetases (氨酰tRNA合成酶)ribosomes (100 proteins and

7、3-4 rRNAs-80%)2022/8/3112Outline Topics 1-4: Four components of translation machinery. T1-mRNA; T2-tRNA; T3-Attachment of amino acids to tRNA (aminoacyl-tRNA synthetases); T4-The ribosomeTopic 5-6: Translation process. T5-initiation; T6-elongation; T7-termination. Topic 8: Translation-dependent regu

8、lation of mRNA and protein stability2022/8/3113Topic 1: mRNAOnly a portion of each mRNA can be translated.The protein-coding region of the mRNA consists of an ordered series of 3-nt-long units called codons that specify the order of amino acids.2022/8/31141-1 polypeptide chains are specified by ORF

9、The protein coding region of each mRNA is composed of a contiguous, non-overlapping string of codons called an opening reading frame (ORF) .Each ORF begins with a start codon and ends with a stop codon.Message RNA2022/8/3115The start codon the first codon of an ORFIn bacteria : AUG, GUG, or UUG (5-3

10、)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 subsequent codons. 2022/8/3116Prokaryotic mRNA (polycistrionic)Eukaryotic mRNA (monocistrionic)2022/8/3117Fig 14-1 Three possible reading

11、frames of the E. coli trp leader sequence2022/8/31181-2 Prokaryotic mRNAs have a ribosome binding site that recruits the translational machineryMessage RNA1-3 Eukaryotic mRNA are modified at their 5 and 3 ends to facilitate translation. 2022/8/3119Ribosome binding site (RBS) or SD-sequence in prokar

12、yotic mRNA, complementary with the sequence at the 3 end of 16S rRNA. Fig 14-2-a structure of prokaryotic mRNA2022/8/3120 Once Kozak sequenceEukaryotic mRNA uses a methylated cap to recruit the ribosome. Once bound, the ribosome scans the mRNA in a 5-3 direction to find the AUG start codon.Kozak seq

13、uence increases the translation efficiency.Poly-A in the 3 end promotes the efficient recycling of ribosomes.Fig 14-2-b2022/8/3121Fig 1-292022/8/3122Topic 2: tRNAAt the heart of protein synthesis is the translation of nucleotide sequence information into amino acids. This work is accomplished by tRN

14、A.2022/8/3123The are many types of tRNA molecules in cell (40).Each tRNA molecule is attached to a specific amino acids (20) and each recognizes a particular codon, or codons (61), in the mRNA.All tRNAs end with the sequence 5-CCA-3 at the 3 end, where the aminoacyl tRNA synthetase adds the amino ac

15、id.2-1: tRNA are adaptors between codons and amino acidsTRANSFER RNA2022/8/3124tRNAs are 75-95 nt in length. There are 15 invariant and 8 semi-invariant residues. The position of invariant and semi-variant nucleosides play a role in either the secondary and tertiary structure. There are many modifie

16、d bases, which sometimes accounting for 20% of the total bases in one tRNA molecule. Over 50 different types of them have been observed.Primary structure2022/8/3125Fig 14-3 unusual basesPseudouridine (U) is a modified base. These modified bases in tRNA lead to improved tRNA function2022/8/3126The cl

17、overleaf structure is 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 RNA2022/8/3127Fig 14-4 the secondary struct

18、ure2022/8/31282-3: tRNAs have an L-shaped 3-D structureFig 14-5 the 3-D structure of tRNAD loopU loop2022/8/3129Formation 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. 2

19、022/8/3130The cloverleaf 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 =pseudouridine) and 8 semi-invariant nucleotides (abbreviations: R, purine; Y, pyrimidine) are indicate

20、d. Optional nucleotides 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、 21, 24, 32, 33, 37, 48, 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/2022/8/3131Base 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 acid a

22、cceptor site at the other (Fig. 14-5). The base pairing is strengthened by base stacking interactions. 2022/8/3132Topic 3: attachment of amino acids to tRNAAmino acids should be attached to tRNA first before adding to polypeptide chain. tRNA molecules to which an amino acid is attached are said to b

23、e charged, and tRNAs lacking an amino acid are said to uncharged. 2022/8/31333-1 tRNAs are charged by attachment of an amino acid to the 3 terminal A of the tRNA via a high energy acyl linkageEnergy: The energy released when the high-energy bond is broken helps drive the peptide bond formation durin

24、g protein synthesis.Enzyme: Aminoacyl tRNA synthetase catalyzing the reaction has three binding sites for ATP, amino acid and tRNA. ATTACHMENT OF AMINO ACIDS TO tRNA2022/8/31343-2 Aminoacyl tRNA synthetases charge tRNA in two steps (reactions)1. Adenylylation (腺苷?；? of amino acids: transfer of AMP t

25、o the COO- end of the amino acids.2. tRNA charging: transfer of the adenylylated amino acids to the 3 end of tRNA, generating aminoacyl-tRNAs (charged tRNA).ATTACHMENT OF AMINO ACIDS TO tRNA2022/8/3135Class I: attach the amino acids to the 2OH of the tRNA, and is usually monomeric.Class II: attach t

26、he amino acids to the 3OH of the tRNA, and is usually dimeric or tetrameric.There are two classes of tRNA synthetases.2022/8/3136Step 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

27、苷酸) intermediate. As a result, the adenylylated aa binds to the synthetase tightly. This step is also called activation of amino acids p652022/8/3137A class II tRNA synthetase2022/8/31382022/8/3139Step 2- tRNA charging: transfer of the adenylated amino acid to the 3 end of the appropriate tRNA via t

28、he 2 or 3-OH group, and the AMP is released as a result.2022/8/3140Nature structural and Molecular Biology, 2005, 12:915-9222022/8/31413-3： each aminoacyl tRNA synthetase attaches a single amino acids to one or more cognate/appropriate tRNAsEach of the 20 amino acids is attached to the appropriate t

29、RNA (s) by aminoacyl-tRNA synthetases.Most amino acids are specified by more than one codon, and by more than one tRNA as well. ATTACHMENT OF AMINO ACIDS TO tRNA2022/8/3142The same synthetase is responsible for charging all tRNAs for a particular amino acid (one synthetaseone amino acid).Consequentl

30、y, most organisms have 20 synthetases for 20 different amino acids.2022/8/31433-4 tRNA synthetases recognize unique structure features of cognate tRNAs ATTACHMENT OF AMINO ACIDS TO tRNAThe recognition has to ensure two levels of accuracy: (1) each tRNA synthetase must recognize the correct set of tR

31、NAs for a particular amino acids; (2) each synthetase must charge all of these isoaccepting tRNAs (即由一種synthetase所識別的不同tRNAs) 2022/8/3144The specificity determinants for accurate recognition are clusters at two distinct sites: the acceptor stem and the anti-codon loop.2022/8/3145Fig 14-8Fig 14-72022

32、/8/3146Identity elements (specificity determinants ) in various tRNA molecules2022/8/31473-5 Aminoacyl-tRNA formation is very accurate: selection of the correct amino acidThe aminoacyl tRNA synthetases discriminate different amino acids according to different natures of their side-chain groups.ATTAC

33、HMENT OF AMINO ACIDS TO tRNA2022/8/3148Fig. 14-92022/8/31493-6 Some aminoacyl tRNA synthetase use an editing pocket to charge tRNAs with high accuracy. ATTACHMENT OF AMINO ACIDS TO tRNA2022/8/3150Isoleucyl tRNA synthetase as an example:Its editing pocket near the catalytic pocket allows it to proof

34、read the product of the adenylation reaction (step #1).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 binding pocket serves as a molecular sieve to exclude AMP-valine etc.ValIle2022/8/3151Therefore, Ile-tR

35、NA 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 100, and the overall selectivity is about 10,000-fold.2022/8/31521. Ribosome recognize tRNAs but not

36、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 correctly or incorrectly charged tRNAs (是否攜帶正確的氨基酸)ATTACHMENT OF AMINO ACIDS TO tRNA2022/8/3153Topic

37、 4: the ribosomeRibosome compositionRibosome cyclePeptide bond formationRibosome structure2022/8/31544-1 the ribosome is composed of a large and a small subunitThe large subunit contains the peptidyl transferase center, which is responsible for the formation of peptide bonds.The small subunit intera

38、cting with mRNA contains the decoding center, in which charged tRNAs read or “decode” the codon units of the mRNA.RIBOSOMES2022/8/3155Fig 14-13* Ribosome2022/8/31564-2: the large and the small subunits undergone association and dissociation during each cycle of translation.RIBOSOMES2022/8/3157Riboso

39、me 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 and dissociation is called the ribosome cycle.2022/8/3158Fig 14-14 Overview of the events of translation/

40、ribosome cycle2022/8/3159Polysome/polyribosome: an mRNA bearing multiple ribosomes Each mRNA can be translated simultaneously by multiple ribosomes Fig 14-15 A polyribosome2022/8/31602022/8/3161RIBOSOMES4-3 New amino acids are attached to the C-terminus of the growing polypeptide chain.Protein is sy

41、nthesized 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.2022/8/3162Fig 14-162022/8/3163The structure of the ribosome 4-5 Ribosomal RNAs are both structural and catalytic determinants of the ribosomes 4-6 Th

42、e 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 structure2022/8/3164Fig 14-17 two views of the ribosome2022/8/31654-6 Three binding site for tRNAsFig 14-18A site: to bind the

43、 aminoacylated-tRNAP-site: to bind the peptidyl-tRNAE-site: to bind the uncharged tRNA 2022/8/3166Fig 14-19 3-D structure of the ribosome including 3 bound tRNA2022/8/31674-7 Channel for mRNA entering and exiting are located in the small subunit (see Fig. 14-18)There is a pronounced kink in the mRNA

44、 between the two codons at P and A sites. This kink places the vacant A site codon for aminoacyl-tRNA interaction. Fig 14-202022/8/31684-7 Channel for polypeptide chain exiting locates in the large subunitThe size of the channel only allow a very limited folding of the newly synthesized polypeptideF

45、ig 14-212022/8/3169Translation processT5: Initiation of translationT6: Elongation of translationT7: termination of translation Watch the animation on your study CD2022/8/3170QuestionsCompare the mechanism of translation initiation in prokaryotes and eukaryotes (similarity and difference)How do amino

46、acyl-tRNA synthetases and the ribosomes contribute to the fidelity of translation, respectively? 2022/8/3171Overview of the events of translationTermination Elongation InitiationFig 14-142022/8/3172T5: Initiation of translation Initiation in prokaryotic cells (1-3)Initiation in eukaryotic cells (4-6

47、)2022/8/31735-1 Prokaryotic mRNAs are initially recruited to the small subunit by base pairing to rRNA.INITIATION OF TRANSLATIONFig 14-23RBS is also called ShineDalgarno sequence2022/8/31745-2 A specialized tRNA (initiator tRNA) charged with a modified methionine (f-Met) binds directly to the prokar

48、yotic small subunit.INITIATION OF TRANSLATIONFig 14-24 2022/8/31755-3 Three initiator factors direct the assembly of an initiation complex that contains mRNA and the initiator tRNA.INITIATION OF TRANSLATIONFormation of the 30S initiation complex: IFs1-3 + 30S + mRNA + fmet-tRNA.2. Formation of the 7

49、0S initiation complex: 50S + 30S + mRNA + fmet-tRNA2022/8/31762022/8/3177Eukaryotic initiation5-4 Eukaryotic ribosomes are recruited to the 5 cap.5-5 The start codon is found by scanning downstream from the 5 end of the mRNA.INITIATION OF TRANSLATIONFigs 14-26 and -272022/8/31785-6 Translation initi

50、ation factors hold eukaryotic mRNAs in circlesTry to explain how the mRNA poly-A tail contributes to the translation efficiency?INITIATION OF TRANSLATIONFig 14-292022/8/3179T6: Translation elongation Aminoacyl-tRNA binding to A sitePeptide bond formationTranslocation2022/8/31806-1 Aminoacyl-tRNAs ar

51、e delivered to the A site by elongation factor EF-TuELONGATION OF TRANSLATIONEF-Tu-GTP binds to aminoacyl-tRNAsDeliver a tRNA to A site on ribosome When correct codon-anticodon occurs, EF-Tu interacts with the factor-binding center on ribosome and hydrolyzes its bound GTPEF-Tu-GDP leaves ribosomeFig

52、 14-302022/8/31816-2 The ribosome uses multiple mechanisms to select against incorrect aminoacyl-tRNAsELONGATION OF TRANSLATIONAdditional hydrogen bonds are formed between two adenine residues of the 16S rRNA and the minor groove of the anticodon-codon pair only when they are correctly paired. Fig 1

53、4-31a2022/8/3182Correct base pairing allows EF-Tu interact with the factor binding center on ribosome, which is important for GTP hydrolysis and EF-Tu release. Fig 14-31b2022/8/3183Only correct base paired aminoacyl-tRNAs remain associated with the ribosome as they rotate into the correct position f

54、or peptide bond formation.Fig 14-31c2022/8/31846-3 Ribosome is a ribozyme (重點，催化如何發(fā)生？)ELONGATION OF TRANSLATIONFig 14-32Fig 14-162022/8/3185ELONGATION OF TRANSLATIONThe role of L27 protein? The role of A2451 nucleotide residue in the 23 rRNA?The role of the 2-OH of the A residue at the 3 of the pept

55、idyl-tRNA (a part of a proton shuttle)? Figure-14-332022/8/31866-4 & 5 Elongation factor EF-G drive translocation of the tRNAs and the mRNA by displacing the tRNA bound to the A site ELONGATION OF TRANSLATION2022/8/3187EF-G mimics a tRNA molecule so as to displace the tRNA bound to the A site EF-G-G

56、DPEF-Tu-GDPNP-Phe-tRNAFig 14-352022/8/31886-6 EF-Tu-GDP and EF-G-GDP must exchange GDP for GTP prior to participating in a new round of elongation.ELONGATION OF TRANSLATIONEF-G-GDP: GDP has a lower affinity, and GDP is released after GTP hydrolysis. The free EF-G rapidly binds a new GTP.EF-Tu-GDP re

57、quires a GTP exchange factor EF-Ts to displace GDP and recruit GTP to EF-Tu. (Fig. 14-36) 2022/8/3189Topic 7: Translation termination Releasing factors act to release the synthesized peptide from the peptidyl tRNA in the ribosome.Ribosome recycling factor, EF-Tu and IF3 act to recycle the ribosome.2

58、022/8/31907-1 & 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 TRANSLATION2022/8/31917-2 Class I releasing factors (bacterial RF1 and RF2, eukaryotic eRF1) recognize stop codons by its pe

59、ptide 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 TRANSLATION2022/8/31927-3 Class II releasing factor remove Class I releasing factor from the

60、 A site. And this function is controlled by GDP/GTP exchange and GTP hydrolysis (Figure 14-39). TERMINATION OF TRANSLATION2022/8/31937-4 Recycling of the ribosome by a combined effort of RRF (ribosome recycling factor), EF-Tu-GTP and IF3. (Figure 14-40)TERMINATION OF TRANSLATIONRRF mimics a tRNA and