1、2022-5-311. Why RNA splicing is important? 2. Chemical reaction: determination of the splice sites, the products, trans-splicing3. Spliceosome: snRNPs, splicing pathway and finding the splice sites4. Self-splicing introns and mechanisms5. Alternative splicing and regulation, alternative spliceosome6

2、. Two different mechanisms of RNA editing7. mRNA transport - a link to translation Key points of the chapter132022-5-32Chapter 14Translation2022-5-33nThis part concerned with one of the greatest challenges in understanding the genehow the gene is expressed.2022-5-34Translation extremely costsnIn rap

3、id growing bacterial cells, protein synthesis consumes a) 80% of the cells energy b) 50% of the cells dry weight.Why?2022-5-35The main challenge of translation nThe genetic information in mRNA cannot be recognized by amino acids. nThe genetic code has to be recognized by an adaptor molecular (transl

4、ator), and this adaptor has to accurately recruit the corresponding amino acid. 2022-5-36 The synthesis of protein molecules using mRNA as the template, in other word, to translate the nucleotide sequence of mRNA into the amino acid sequence of protein according to the genetic codon.Translation2022-

5、5-37Translation 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%)2022-5-38Outline nTopics 1: Four components of translation machinery. mRNA; tRNA; Attachment of amino acids to tRNA (aminoacyl-tRNA syntheta

6、ses); The ribosomenTopic 2: Translation process. initiation; elongation; termination. nTopic 3: Translation-dependent regulation of mRNA and protein stability2022-5-39Topic 1-1: mRNAnOnly a portion of each mRNA can be translated.nThe protein-coding region of the mRNA consists of an ordered series of

7、 3-nt-long units called codons that specify the order of amino acids.2022-5-310polypeptide 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) .nAn ORF should begins with a start codon

8、 and end with stop codon.nmRNA containing more than one ORF is called polycistronic mRNAs.Message RNA2022-5-311The 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 poly

9、peptide chain.2.Defines the reading frame for all subsequent codons. 2022-5-312Fig 14-1 Three possible reading frames of the E. coli trp leader sequence.2022-5-313Prokaryotic mRNAs have a ribosome binding site (RBS) that recruits the translational machineryEukaryotic mRNA are modified at their 5 and

10、 3 ends to facilitate translation. 2022-5-314nRBS or SD-sequence in prokaryotic mRNA, complementary with the sequence at the 3 end of 16S rRNA. Fig 14-2-a structure of mRNA2022-5-315 Once Kozak sequencenEukaryotic mRNA uses a methylated cap to recruit the ribosome. Once bound, the ribosome scans the

11、 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-b2022-5-316nThe protein coding region of each mRNA is The protein coding region of each mRNA is composed of a contiguo

12、us, non-overlapping string composed of a contiguous, non-overlapping string of codons called an of codons called an opening reading frameopening reading frame ( (ORFORF) .) .nEach ORF begins with a Each ORF begins with a start codonstart codon and ends and ends with a with a stop codonstop codon. .n

13、Polycistrionic (Prokaryotic mRNA )nmonocistrionic (Eukaryotic mRNA )2022-5-3171-2: tRNAAt the heart of protein synthesis is the translation of nucleotide sequence information into amino acids. This work is accomplished by tRNA.2022-5-318nThe are many types of tRNA The are many types of tRNA molecule

14、s in cell molecules in cell (40)(40). .nEach tRNA molecule is attached to a Each tRNA molecule is attached to a specific amino acids specific amino acids (20)(20) and each and each recognizes a particular codon, or recognizes a particular codon, or codons codons (61)(61), in the mRNA., in the mRNA.n

15、All tRNAs end with the sequence 5-All tRNAs end with the sequence 5-CCA-3 at the CCA-3 at the 3 end3 end, where the , where the aminoacyl tRNA synthetase adds the aminoacyl tRNA synthetase adds the amino acid.amino acid.tRNA are adaptors between codons and amino acids2022-5-319ntRNAs are 75-95 nt in

16、 length. nThere 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. nThere are many modified bases, which sometimes accounting for 20% of the total bases in one tRNA molecule. Over 50 diffe

17、rent types of them have been observed.Primary structure2022-5-320Fig 14-3 unusual basesnPseudouridine ( U) is a modified base. These modified bases in tRNA lead to improved tRNA function.2022-5-321nThe cloverleaf structure is a common secondary structural representation of tRNA molecules which shows

18、 the base paring of various regions to form four stems (arms) and three loops. tRNAs share a common secondary structure that resemble a cloverleaf2022-5-322Fig 14-4 The secondary structure2022-5-323xtRNA secondary structure:The cloverleaf structure Variable loopY: pyrimidineR: purine2022-5-3247 base

19、 pairs between 5-and 3-end form the amino acid acceptor arm which has no loop. 2022-5-325Composed of 3 or 4 bp stem and a loop called the D-loop (DHU-loop) usually containing the modified base dihydrouracil. （二氫尿嘧啶）二氫尿嘧啶）2022-5-326n Consisting of a 5 bp stem and a 7 residues loop .n anticodon: three

20、 adjacent nucleosides, at positions 3436 in a tRNA molecular, that basepair with a codon sequence in a mRNA molecular.2022-5-327Variable loop 35(class I) /1321(class II) residues.T-arm Named after the sequence TC. 2022-5-328tRNAs have an L-shaped threedimentional structure2022-5-329n9 hydrogen bones

21、 (tertiary hydrogen bones) help the formation of tRNA tertiary structure, mainly involving in the base paring between the invariant bases.nBase 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 acceptor site at

22、the other . nThe base pairing is strengthened by base stacking interactions. Formation:2022-5-3301-3: attachment of amino acids to tRNAnAmino acids should attach to tRNA first before adding to polypeptide chain. ntRNA molecules to which an amino acid is attached are said to be charged, and tRNAs lac

23、king an amino acid are said to uncharged. 2022-5-331tRNAs 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 during protein synthesis.nEnzyme:

24、 catalyzing the reaction has three binding sites for ATP, amino acid and tRNA. 2022-5-332Aminoacyl tRNA synthetases charge tRNA in two steps1. Adenylylation (腺苷酰化腺苷?；? of amino acids: transfer of AMP to the COO- end of the amino acids.2. tRNA charging: transfer of the adenylylated amino acids to the

25、 3 end of tRNA, generating aminoacyl-tRNAs (charged tRNA).2022-5-333Step 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 activation of a

26、mino acidsFig 14-6a2022-5-334Step 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.Fig 14-6b2022-5-335nClass I: attach the amino acids to the 2OH of the tRNA, and is usually monomeric.nClass II:

27、attach the amino acids to the 3OH of the tRNA, and is usually dimeric or tetrameric.There are two classes of tRNA synthetases.2022-5-336Each 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-tR

28、NA synthetases.nMost amino acids are specified by more than one codon, and by more than one tRNA as well. 2022-5-337nThe 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

29、 amino acids.2022-5-338tRNA synthetases recognize unique structure features of cognate tRNAs nThe 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 (即由一種即由一種synthetase所識(shí)別的不同所識(shí)別的

30、不同tRNAs) 2022-5-339nThe specificity determinants for accurate recognition are clusters at two distinct sites: the acceptor stem and the anti-codon loop.2022-5-340Fig 14-8Fig 14-72022-5-341Identity elements (specificity determinants ) in various tRNA molecules 2022-5-342Aminoacyl-tRNA formation is ve

31、ry accurate: selection of the correct amino acidnThe aminoacyl tRNA synthetases discriminate different amino acids according to different natures of their side-chain groups.2022-5-343Fig 14-9 Distinguish features of similar amino acid.氨基酰氨基酰tRNA合成酶可以通合成酶可以通過氨基酸在過氨基酸在體積、構(gòu)型和化體積、構(gòu)型和化學(xué)組成上的不同學(xué)組成上的不同來正確選擇

32、來正確選擇相應(yīng)的氨基酸。相應(yīng)的氨基酸。有些則通過利用有些則通過利用編輯口袋編輯口袋來高精度地負(fù)載來高精度地負(fù)載tRNA。一旦錯(cuò)誤的負(fù)載，則核糖一旦錯(cuò)誤的負(fù)載，則核糖體不能辨別，即核糖體接受體不能辨別，即核糖體接受任一由任一由密碼子密碼子-反密碼子反密碼子相互相互作用而形成的負(fù)載作用而形成的負(fù)載tRNA。2022-5-344Proofreading Proofreading occurs at step 2 when a synthetase carries out step 1 of the aminoacylation reaction with the wrong, but chemica

33、lly similar, amino acid. nSome enzymes have editing pocket to do proofreading by matching the wrong product and hydrolyzing it .nSynthetase will not attach the aminoacyl adenylate to the cognate tRNA, but hydrolyze the aminoacyl adenylate instead.2022-5-345Some aminoacyl tRNA synthetase use an editi

34、ng pocket to charge tRNAs with high accuracy. 2022-5-346Isoleucyl 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 hydr

35、olyzed. 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.ValIle2022-5-347Therefore, Ile-tRNA synthetase discriminates against valine twice: the initial binding and adenylylation of the amino acidthe editing of the adenylylate

36、d amino acid. Each step discriminates by a factor of 100, and the overall selectivity is about 10,000-fold.2022-5-3481. 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 t

37、RNA. Ribosomes is unable to discriminate between correctly or incorrectly charged tRNAs (是否攜帶正確的氨基酸)2022-5-349Fig 14-10 Cysteinyl-tRNA charged with C or A半光氨酰tRNA丙氨酰tRNA2022-5-350氨酰tRNA合成酶的底物及選擇特異性tRNA氨基酸ATP氨基酸接受臂反密碼子環(huán)其他的決定區(qū)側(cè)鏈基團(tuán)體積、結(jié)構(gòu)和化學(xué)組組成上的差異催化口袋編輯口袋一個(gè)氨酰tRNA合成酶對(duì)應(yīng)一個(gè)特定的氨基酸和一組同工tRNA2022-5-3511-4: the

38、ribosome1.Ribosome composition2.Ribosome cycle3.Peptide bond formation4.Ribosome structure核糖體是由一大一小兩個(gè)亞基結(jié)合形成，主要成分是相互纏繞的核糖體是由一大一小兩個(gè)亞基結(jié)合形成，主要成分是相互纏繞的rRNA和核糖體蛋白質(zhì)。核糖體是細(xì)胞內(nèi)蛋白質(zhì)合成的場所，能讀取和核糖體蛋白質(zhì)。核糖體是細(xì)胞內(nèi)蛋白質(zhì)合成的場所，能讀取mRNA所含的遺傳信息，并使之轉(zhuǎn)化為蛋白質(zhì)中氨基酸的序列信息所含的遺傳信息，并使之轉(zhuǎn)化為蛋白質(zhì)中氨基酸的序列信息以合成蛋白質(zhì)。在原核生物及真核生物的細(xì)胞中都有核糖體存在。以合成蛋白質(zhì)。在原核生

39、物及真核生物的細(xì)胞中都有核糖體存在。2022-5-3521-4-1 The ribosome is composed of a large and a small subunitnThe large subunit contains the peptidyl transferase center (肽基轉(zhuǎn)移酶中心肽基轉(zhuǎn)移酶中心), which is responsible for the formation of peptide bonds.nThe small subunit interacting with mRNA contains the decoding center(解碼解碼中心中

40、心), in which charged tRNAs read or “decode” the codon units of the mRNA.2022-5-353Fig 14-13* Ribosome2022-5-354nRibosome cycles: During the protein systhesis, the small and large ribosome subunits associate with each other and the mRNA, translate the target mRNA, and then dissociate after each round

41、 of translation. 1-4-2 The large and the small subunits undergone association and dissociation during each cycle of translation.2022-5-355Fig 14-14 Overview of the events of translation：The ribosome cycle.2022-5-356Polysome/polyribosome: an mRNA bearing multiple ribosomesFig 14-15 A polyribosome2022

42、-5-3572022-5-3581-4-3 New amino acids are attached to the C-terminus of the growing polypeptide chain.Protein is synthesized in a N- to C- terminal directionPeptide bonds are formed by transfer of the growing peptide chain from peptidyl- tRNA to aminoacyl-tRNA.2022-5-359Fig 14-16氨基酰氨基酰tRNA的氨基基的氨基基團(tuán)攻

43、擊肽基酰團(tuán)攻擊肽基酰tRNA上上多肽多肽C端的羧基基團(tuán)，端的羧基基團(tuán)，形成一個(gè)新的肽鍵：形成一個(gè)新的肽鍵：1）這種肽鍵的形成方）這種肽鍵的形成方式?jīng)Q定蛋白質(zhì)的式?jīng)Q定蛋白質(zhì)的N端先端先于于C端的合成；端的合成；2）多肽鏈從肽酰）多肽鏈從肽酰tRNA轉(zhuǎn)移到氨酰轉(zhuǎn)移到氨酰tRNA上。上。因此，新肽鍵形成的反因此，新肽鍵形成的反應(yīng)稱為應(yīng)稱為肽轉(zhuǎn)移酶反應(yīng)肽轉(zhuǎn)移酶反應(yīng)。2022-5-3601-4-4 The structure of the ribosome Ribosomal RNAs are both structural and catalytic determinants of the ribos

44、omes The ribosome has three binding sites for tRNA (A site, P site and E site). Channels through the ribosome allow the mRNA and growing polypeptide to enter and/or exit the ribosome.2022-5-361Ribosome structureFig 14-17 two views of the ribosometRNA結(jié)合結(jié)合的位點(diǎn)的位點(diǎn)2022-5-362Three binding site for tRNAsFi

45、g 14-18A site: to bind the aminoacylated-tRNAP-site: to bind the peptidyl-tRNAE-site: to bind the uncharged tRNA 2022-5-363Fig 14-19 3-D structure of the ribosome including 3 bound tRNA黃黃/紅紅/綠：與核糖綠：與核糖體體E/P/A位點(diǎn)結(jié)合位點(diǎn)結(jié)合的的tRNA肽酰轉(zhuǎn)移酶中心：肽酰轉(zhuǎn)移酶中心：新合成的多肽鏈從新合成的多肽鏈從此中心離開此中心離開解碼中心：只允許解碼中心：只允許單鏈的單鏈的mRNA進(jìn)入進(jìn)入進(jìn)出通道之間

46、進(jìn)出通道之間有一個(gè)有一個(gè)tRNA可進(jìn)入的區(qū)域可進(jìn)入的區(qū)域2022-5-364Channel for mRNA entering and exiting are located in the small subunit mRNA的兩個(gè)密碼子之間存的兩個(gè)密碼子之間存在明顯的扭結(jié)在明顯的扭結(jié)(kink)，有利，有利于維持正確的可讀框。另外，于維持正確的可讀框。另外，扭結(jié)使核糖體易位后留在空扭結(jié)使核糖體易位后留在空著的著的A位點(diǎn)，阻止隨后進(jìn)入的位點(diǎn)，阻止隨后進(jìn)入的氨基酰氨基酰-tRNA與比鄰該密碼與比鄰該密碼子的堿基相接觸。子的堿基相接觸。Fig 14-20EPA2022-5-365Channel f

47、or polypeptide chain exiting locates in the large subunitThe size of the channel only allow a very limited folding of the newly synthesized polypeptide。Fig 14-212022-5-366The role of ribosome in protein synthesislRibosomes coordinate protein synthesis by placing the mRNA, aminoacyl-tRNAs and associa

48、ted protein factors in their correct positions relative to one another.lb. Components of ribosomes, including the rRNAs, catalyze at least some of the chemical reactions occurring during translation. Ribosomes components: Ribosomes RNA Ribosomes protein2022-5-367Topic 2: Translation process Initiati

49、on of translation Elongation of translation Termination of translation2022-5-368Overview of the events of translationTermination Elongation InitiationFig 14-142022-5-3692-1: Initiation Initiation in prokaryotic cells Initiation in eukaryotic cells2022-5-370Initiation. The ribosome must be recruited

50、to the mRNA. A charged tRNA must be placed into the P site of the ribosome. the ribosome must be positioned over the start codonA specialized tRNA charged with a modified methionine binds directly to the prokaryotic small subunit.Prokaryotic mRNA are initially recrutied to the small subunit by base-

51、paring to 16s rRNA Eukaryotic ribosomes are recruited to the mRNA by the 5 cap.2022-5-371Prokaryotic mRNAs are initially recruited to the small subunit by base pairing to rRNA.Fig 14-23RBS is also called ShineDalgarno sequence2022-5-372A specialized tRNA (initiator tRNA) charged with a modified meth

52、ionine (f-Met) binds directly to the prokaryotic small subunit.Fig 14-24 2022-5-373Three initiator factors direct the assembly of an initiation complex that contains mRNA and the initiator tRNA.1. Formation of the 30S initiation complex: IFs1-3 + 30S + mRNA + fmet-tRNA.2. Formation of the 70S initia

53、tion complex: 50S + 30S + mRNA + fmet-tRNAIF：translation initiation factor2022-5-374Initiation Factors and the Initiation ComplexnIF1 prevents tRNA from binding to small subunit prematurely.nIF2 is a GTPase that interacts with small subunit, IF1 and the charged tRNA-fMet.nIF3 binds small subunit and

54、 prevents premature binding to large subunit.2022-5-375Figure 14-25 Prokaryotic Initiation Process2022-5-376Topic 2-1-2 Eukaryotic initiationn Eukaryotic ribosomes are recruited to the 5 cap.n The start codon is found by scanning downstream from the 5 end of the mRNA.n Initiation of translation in b

55、oth eukaryotes and prokaryotes, 1) use a start codon and initiator tRNA; 2) use initiation factors to form a complex with the small ribosomal subunit.2022-5-377Figure 14-261. 在真核細(xì)胞中，起始在真核細(xì)胞中，起始tRNA與核糖體小亞基首與核糖體小亞基首先結(jié)合，然后在先結(jié)合，然后在mRNA上上以以5到到3方向方向掃描掃描并并尋找第一個(gè)尋找第一個(gè)AUG序列。序列。2. 另一組輔助因子介導(dǎo)對(duì)另一組輔助因子介導(dǎo)對(duì)mRNA的識(shí)別。的

56、識(shí)別。3. 核糖體的大亞基在起始核糖體的大亞基在起始tRNA與起始密碼子堿基與起始密碼子堿基配對(duì)之后被募集。配對(duì)之后被募集。48S preinitiation complex2022-5-378Figure 14-27 Identification of the initiating AUG by the eukaryotic small ribosomal subunit.80S preinitiation complex2022-5-379Translation initiation factors hold eukaryotic mRNAs in circlesTry to explai

57、n how the mRNA poly-A tail contributes to the translation efficiency?Fig 14-282022-5-380.The correct aminoacyl-tRNA is loaded into the A site of the ribosome.a peptide bond is formed between the aminoacyl-tRNA in the A site and the peptide chain that is attached to the peptidyl-tRNA in the P site.th

58、e resulting peptidyl-tRNA in the A site and its associated codon must be translocated to the P site. Topic 2-2: Translation elongation Fig 14-292022-5-381Aminoacyl-tRNAs are delivered to the A site by elongation factor EF-TuFig 14-302022-5-382The ribosome uses multiple mechanisms to select against i

59、ncorrect aminoacyl-tRNAsnThe error rate of translation is between 10-3 to 10-4.nThe ultimate basis for the selection of the correct aminoacyl- tRNA is the base pairing between the charged tRNA and the codon displayed in the A site of the ribosome.nThere are three mechanisms to ensure correct pairing

60、 between the tRNA and the RNA.2022-5-383Mechanism 1：The fidelity of codon recognition involves two adjacent adenine residues in the 16s rRNA component located within the A site.Fig 14-31a2022-5-384Fig 14-31bMechanism 22022-5-385Fig 14-31cMechanism 32022-5-386Ribosome is a ribozyme Fig 14-32一旦正確的負(fù)載一旦