Presentation on theme: "Section III: Expression of the Genome"— Presentation transcript:
1 Section III: Expression of the Genome Mechanisms of transcriptionRNA splicingTranslationThe genetic code
2 Mechanisms of Transcription chapter12:mechanisms of transcription2017/4/15Molecular Biology CourseMechanisms of TranscriptionRNA polymerase and transcription cycleThe transcription cycle in bacteriaTranscription in eukaryotes
3 The Central Dogma DNA RNA PROTEIN Transcription Translation replication
4 Transcription is very similar to DNA replication but there are some important differences: RNA is made of ribonucleotidesRNA polymerase catalyzes the reactionThe synthesized RNA does not remain base-paired to the template DNA strandLess accurate (error rate: 10-4)
5 Transcription selectively copies only certain parts of the genome and makes one to several hundred, or even thousand, copies of any given section of the genome. (Replication?)
6 Transcription of DNA into RNA Transcription bubbleTranscription of DNA into RNA
7 Topic 1: RNA Polymerase and The Transcription Cycle Mechanisms of TranscriptionTopic 1: RNA Polymerase and The Transcription Cycle
8 RNA polymerases come in different forms, but share many features RNA polymerase and the transcription cycleRNA polymerases come in different forms, but share many featuresRNA polymerases performs essentially the same reaction in all cellsBacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and III
9 RNA Pol II is the focus of eukaryotic transcription, because it is the most studied polymerase, and is also responsible for transcribing most genes-indeed, essentially all protein-encoding genesRNA Pol I transcribe the large ribosomal RNA precursor geneRNA Pol II transcribe tRNA gene, some small nuclear RNA genes and the 5S rRNA genes
11 The bacterial RNA polymerase The core enzyme alone synthesizes RNAb’abaw
12 The same color indicate the homologous of the two enzymes a w prokaryoticb’RNAP ComparisonabThe same color indicate the homologous of the two enzymesaweukaryoticRPB2RPB3RPB1RPB11RPB6
13 “Crab claw” shape of RNAP (The shape of DNA pol is__) Active center cleft
14 There are various channels allowing DNA, RNA and ribonucleotides (rNTPs) into and out of the enzyme’s active center cleft
15 Transcription by RNA polymerase proceeds in a series of steps RNA polymerase and the transcription cycleTranscription by RNA polymerase proceeds in a series of stepsInitiationElongationTermination
16 InitiationPromoter: the DNA sequence that initially binds the RNA polymeraseThe structure of promoter-polymerase complex undergoes structural changes to proceed transcriptionDNA at the transcription site unwinds and a “bubble” formsDirection of RNA synthesis occurs in a 5’-3’ direction (3’-end growing)
18 ElongationOnce the RNA polymerase has synthesized a short stretch of RNA (~ 10 nt), transcription shifts into the elongation phase.This transition requires further conformational change in polymerase that leads it to grip the template more firmly.Functions: synthesis RNA, unwinds the DNA in front, re-anneals it behind, dissociates the growing RNA chain
19 TerminationAfter the polymerase transcribes the length of the gene (or genes), it will stop and release the RNA transcript.In some cells, termination occurs at the specific and well-defined DNA sequences called terminators. Some cells lack such termination sequences.
21 Transcription initiation involves 3 defined steps RNA polymerase and the transcription cycleTranscription initiation involves 3 defined stepsForming closed complexForming open complexPromoter escape
22 Closed complex The initial binding of polymerase to a promoter DNA remains double strandedThe enzyme is bound to one face of the helix
23 Open complexthe DNA strand separate over a distance of ~14 bp (-11 to +3 ) around the start site (+1 site)Replication bubble forms
24 Stable ternary complex RNA polymerase and the transcription cycleStable ternary complexThe enzyme escapes from the promoterThe transition to the elongation phaseStable ternary complex=DNA +RNA + enzyme
25 Topic 2 The transcription cycle in bacteria Mechanisms of TranscriptionTopic 2 The transcription cycle in bacteria
26 The transcription cycle in bacteria Bacterial promoters vary in strength and sequences, but have certain defining features
27 Holoenzyme= factor + core enzyme In cell, RNA polymerase initiates transcription only at promoters. Who confers the polymerase binding specificity?,
28 Promoters recognized by E. coli s factor The predominant s factor in E. coli is s70.Promoter recognized by s70 contains two conserved sequences (-35 and –10 regions/elements) separated by a non-specific stretch of nt.Position +1 is the transcription start site.
29 bacterial promoterThe distance is conserveds70 promoters contain recognizable –35 and –10 regions, but the sequences are not identical.Comparison of many different promoters derives the consensus sequences reflecting preferred –10 and –35 regions
31 Promoters with sequences closer to the consensus are generally “stronger” than those match less well. (What does “stronger” mean?)The strength of the promoter describes how many transcripts it initiates in a given time.
32 bacterial promoterConfers additional specificityUP-element is an additional DNA elements that increases polymerase binding by providing the additional interaction site for RNA polymerase
33 bacterial promoterAnother class of s70 promoter lacks a –35 region and has an “extended –10 element” compensating for the absence of –35 region
34 The s factor mediates binding of polymerase to the promoter The transcription cycle in bacteriaThe s factor mediates binding of polymerase to the promoterThe s70 factor comprises four regions called s region 1 to s region 4.
35 regions of sRegion 4 recognizes -35 element Region 2 recognizes -10 elementRegion 3 recognizes the extended –10 element
36 Binding of –35 Two helices within region 4 form a common DNA-binding motif, called a helix-turn-helix motifOne helix inserts into the DNA major groove interacting with the bases at the –35 region. The other helix lies across the top of the groove, contacting the DNA backboneHelix-turn-helix DNA-binding motif
37 Interaction with –10 is more elaborate (精细) and less understood The -10 region is within DNA melting regionThe a helix recognizing –10 can interacts with bases on the non-template strand to stabilize the melted DNA.
38 UP-element is recognized by a carboxyl terminal domain of the a-subunit (aCTD), but not by s factor s and a subunits recruit RNA pol core enzyme to the promoter
39 This transition is called Isomerization (异构化) The transcription cycle in bacteriaTransition to the open complex involves structural changes in RNA polymerase and in the promoter DNAThis transition is called Isomerization (异构化)
40 For s70 –containing RNA polymerase, isomerization is a spontaneous conformational change in the DNA-enzyme complex to a more energetically favorable form. (No extra energy requirement)
41 Change of the promoter DNA the opening of the DNA double helix, called “melting”, at positions -11 and +3.
42 The striking structural change in the polymerase 1. the b and b’ pincers down tightly on the downstream DNA2. A major shift occurs in the N-terminal region of s (region 1.1) shifts. In the closed complex, s region 1.1 is in the active center; in the open complex, the region 1.1 shift to the outside of the center, allowing DNA access to the cleft
43 channels into and out of the open complex NTP uptake channel is in the backDNA entering channelchannels into and out of the open complex
44 The transcription cycle in bacteria Transcription is initiated by RNA polymerase without the need for a primerInitiation requires:The initiating NTP (usually an A) is placed in the active siteThe initiating ATP is held tightly in the correct orientation by extensive interactions with the holoenzyme
45 The transcription cycle in bacteria RNA polymerase synthesizes several short RNAs before entering the elongation phaseAbortive initiation: the enzyme synthesizes and releases short RNA molecules less than 10 nt.
46 Structural barrier for the abortive initiation The 3.2 region of s factor lies in the middle of the RNA exit channel in the open complex.Ejection of this region from the channel (1) is necessary for further RNA elongation, (2) takes the enzyme several attempts
47 channels into and out of the open complex NTP uptake channel is in the backDNA entering channelchannels into and out of the open complex
48 The transcription cycle in bacteria The elongating polymerase is a processive machine that synthesizes and proofreads RNA
49 Synthesizing by RNA polymerase DNA enters the polymerase between the pincersStrand separation in the catalytic cleftNTP additionRNA product spooling out (Only 8-9 nts of the growing RNA remain base-paired with the DNA template at any given time)DNA strand annealing in behind
50 Proofreading by RNA polymerase Pyrohosphorolytic （焦磷酸键解）editing: the enzyme catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi.Hydrolytic （水解）editing: the enzyme backtracks by one or more nucleotides and removes the error-containing sequence. This is stimulated by Gre factor, a elongation stimulation factor.
51 Transcription is terminated by signals within the RNA sequence The transcription cycle in bacteriaTranscription is terminated by signals within the RNA sequenceTerminators: the sequences that trigger the elongation polymerase to dissociate from the DNARho-dependent (requires Rho protein)Rho-independent, also called intrinsic (内在) terminator
52 Rho-independent terminator contains a short inverted repeat (~20 bp) and a stretch of ~8 A:T base pairs.
53 transcription termination Weakest base pairing: A:Umake the dissociation easier
54 Rho (r) -dependent terminators Have less well-characterized RNA elements, and requires Rho protein for terminationRho is a ring-shaped single-stranded RNA binding protein, like SSBRho binding can wrest (夺取) the RNA from the polymerase-template complex using the energy from ATP hydrolysisRho binds to rut (r utilization) RNA sitesRho does not bind the translating RNA
55 the r transcription terminator RNA tread trough the “ring”Hexamer,Open ring
56 Topic 3: transcription in eukaryotes Mechanisms of TranscriptionTopic 3: transcription in eukaryotes
57 Comparison of eukaryotic and prokaryotic RNA polymerases Eukaryotes: Three polymerase transcribes different class of genes: Pol I-large rRNA genes; Pol II-mRNA genes; Pol III- tRNA, 5S rRNA and small nuclear RNA genes (U6)Prokaryotes: one polymerase transcribes all genes
58 Comparison of eukaryotic and prokaryotic promoter recognition Eukaryotes: general transcription factors (GTFs). TFI factors for RNAP I, TFII factors for RNAP II and TFIII factors for RNAP IIIProkaryotes: s factors
59 In addition to the RNAP and GTFs, in vivo transcription also requires Mediator complexDNA-binding regulatory proteinschromatin-modifying enzymesWhy??
60 The transcription in eukaryotes RNA polymerase II core promoters are made up of combinations of 4 different sequence elementsEukaryotic core promoter (~40 nt): the minimal set of sequence elements required for accurate transcription initiation by the Pol II machinery in vitro
61 Pol II core promoter TFIIB recognition element (BRE) The TATA element/boxInitiator (Inr)The downstream promoter element (DPE)
62 Regulatory sequencesThe sequence elements other than the core promoter that are required to regulate the transcription efficiencyThose increasing transcription:Promoter proximal elementsUpstream activator sequences (UASs)EnhancersThose repressing elements: silencers, boundary elements, insulators (绝缘体)
63 RNA Pol II forms a pre-initiation complex with GTFs at the promoter The transcription in eukaryotesRNA Pol II forms a pre-initiation complex with GTFs at the promoterThe involved GTFIIs (general transcription factor for Pol II)TFIID=TBP (TATA box binding protein) + TAFs (TBP association factors)TFIIA, B, F, E, H
64 TBP in TFIID binds to the TATA box TFIIA and TFIIB are recruited with TFIIB binding to the BRERNA Pol II-TFIIF complex is then recruitedTFIIE and TFIIH then bind upstream of Pol II to form the pre-initiation complexPromoter melting using energy from ATP hydrolysis by TFIIH )Promoter escapes after the phosphorylation of the CTD tail
65 Promoter escapeStimulated by phosphorylation of the CTD (C-terminal domain) tail of the RNAP IICTD contains the heptapeptide repeat Tyr-Ser-Pro-Thr-Ser-Pro-SerPhosphorylation of the CTD “tail” is conducted by a number of specific kinases including a subunit of TFIIH
66 The transcription in eukaryotes TBP binds to and distorts DNA using a b sheet inserted into the minor grooveUnusual (P367 for the detailed mechanism)The need for that protein to distort the local DNA structure
67 A:T base pairs (TATA box) are favored because they are more readily distorted to allow initial opening of the minor groove
68 The other GTFs also have specific roles in initiation The transcription in eukaryotesThe other GTFs also have specific roles in initiation~ 10 TAFs: (1) two of them bind DNA elements at the promoter (Inr and DPE); (2) several are histone-like TAFs and might bind to DNA similar to that histone does; (3) one regulates the binding of TBP to DNA
69 TFIIB: (1) a single polypeptide chain, (2) asymmetric binding to TBP and the promoter DNA (BRE), (3)bridging TBP and the polymerase, (4) the N-terminal inserting in the RNA exit channel resembles the s3.2 .TFIIB-TBP-promoter complex
70 TFIIF: (1) a two subunit factor, (2) binding of Pol II-TFIIF stabilizes the DNA-TBP-TFIIB complex, which is required for the followed factor bindingTFIIE: recruits and regulates TFIIHTFIIH: (1) controls the ATP-dependent transition of the pre-initiation complex to the open complex, (2) contains 9 subunits and is the largest GTF; two functions as ATPase and one is protein kinase. (3) important for promoter melting and escape. (4) ATPase functions in nucleotide mismatch repair, called transcription-coupled repair.
71 in vivo, transcription initiation requires additional proteins The transcription in eukaryotesin vivo, transcription initiation requires additional proteinsThe mediator complexTranscriptional regulatory proteinsNucleosome-modifying enzymesTo counter the real situation that the DNA template in vivo is packed into nucleosome and chromatin
72 assembly of the pre-initiation complex in presence of mediator, nucleosome modifiers and remodelers, and transcriptional activators
73 Mediator consists of many subunits, some conserved from yeast to human The transcription in eukaryotesMediator consists of many subunits, some conserved from yeast to humanMore than 20 subunits7 subunits show significant sequence homology between yeast and humanOnly subunit Srb4 is essential for transcription of essentially all Pol II genes in vivoOrganized in modules (模块)
75 Pol II + mediator + some of GTFs core polymerase + s factor Eukaryotic RNA Pol II holoenzyme is a putative preformed complex:Pol II + mediator + some of GTFsProkaryotic RNA Polymerase holoenzyme:core polymerase + s factor
76 A new set of factors stimulate Pol II elongation and RNA proofreading The transcription in eukaryotesA new set of factors stimulate Pol II elongation and RNA proofreading
77 Transition from the initiation to elongation involves the Pol II enzyme shedding (摆脱) most of its initiation factors (GTF and mediators) and recruiting other factors:(1) Elongation factors: factors that stimulate elongation, such as TFIIS and hSPT5.(2) RNA processing (RNA 加工) factorsRecruited to the C-terminal tail of the CTD of RNAP II to phosphorylate the tail for elongation stimulation, proofreading, and RNA processing like splicing and polyadenylation.
78 RNA processing enzymes are recruited by the tail of polymerase
79 Some elongation factors P-TEFb:phosphorylates CTDActivates hSPT5Activates TAT-SF1TFIIS:Stimulates the overall rate of elongation by resolving the polymerase pausingProofreading
80 The transcription in eukaryotes Elongation polymerase is associated with a new set of protein factors required for various types of RNA processingRNA processing:Capping of the 5’ end of the RNASplicing of the introns (most complicated)Poly adenylation (多聚腺苷化) of the 3’ end
81 Elongation, termination of transcription, and RNA processing are interconnected/ coupled (偶联的) to ensure the coordination (协同性) of these eventsEvidence: this is an overlap in proteins involving in those eventsThe elongation factor hSPT5 also recruits and stimulates the 5’ capping enzymeThe elongation factor TAT-SF1 recruits components for splicing
82 Function of poly(A) tail Increased mRNA stabilityIncreased translational efficiencySplicing of last intronAAAAAA
83 Function of 5´cap Protection from degradation Increased translational efficiencyTransport to cytoplasmSplicing of first intron
84 RNA processing 1 5’ end capping The “cap”: a methylated guanine joined to the RNA transcript by a 5’-5’ linkageThe linkage contains 3 phosphates3 sequential enzymatic reactionsOccurs early
85 Splicing: joining the protein coding sequences Dephosphorylation of Ser5 within the CTD tail leads to dissociation of capping machineryFurther phosphorylation of Ser2 recruits the splicing machinery
86 3’ end polyadenylation Linked with the termination of transcription The CTD tail is involved in recruiting the polyadenylation enzymesThe transcribed poly-A signal triggers the reactionsCleavage of the messageAddition of poly-ATermination of transcription
87 polyadenylation and termination 1. CPSF (cleavage and polyadenylation specificity factor) & CstF (cleavage stimulation factor) bind to the poly-A signal, leading to the RNA cleavage2. Poly-A polymerase (PAP) adds ~ 200 As at the 3’ end of the RNA, using ATP as a substratepolyadenylation and termination
89 Models to explain the link between polyadenylation and termination (see the animation on your CD) Model 1: The transfer of the 3’ processing enzyme to RNAP II induces conformational change—RNAP II processivity reduces—spontaneous terminationModel 2: absence of a 5’cap on the second RNA molecule—recognized by the RNAP II as improper—terminate transcription
90 RNA Pol I & III recognize distinct promoters , using distinct sets of transcription factors, but still require TBPThe transcription in eukaryotesPol I: transcribes rRNA precursor encoding gene (multi-copy gene)Pol III: transcribes tRNA genes, snRNA genes and 5S rRNA genes
91 Pol I promoter recognition Upstream control elementUBF binds to the upstream of UCE, bring SL1 to the downstream part of UCE. SL1 in turn recruits RNAP I to the core promoter for transcriptionPol I promoter region
92 Pol III promoter recognition 1. Different forms, 2 Pol III promoter recognition 1. Different forms, 2. locates downstream of the transcription siteTFIIIC binds to the promoter, recruiting TFIIIB, which in turn recruits RNAP IIIPol III core promoter
93 Key points of the chapter RNA polymerases (RNAP, 真核和原核的异同) and transcription cycle (Initiation is more complicate, details in bacteria)Transcription cycle in bacteria:(1) promoters (elements), s factor (4 domains), aCTD, abortive initiation (why?)(2) Structures accounting for formation of the closed complex, transitions to open complex and then stable ternary complex.(3) Elongation and editing by polymerase (10-4)(4) Termination: Rho-independent and Rho-dependent mechanism
94 Transcription cycle in eukaryotes: Promoters (elements), general transcription factors (GTF),RNAP II transcription---the roles of GTFs and the CTD tail of RNAP II in promoter recognition, formation of the pre-initiation complex, promoter melting, promoter escape---in vivo requires mediator complex, nucleosome modifying enzymes and transcription regulatory proteins.---elongation and proofreading involve a new set of GTFs (What)---coupled with RNA processing (How)
95 RNAP I and III transcription ---GTFs and promoter recognition, formation of the initiation complex
98 Most of the eukaryotic genes are mosaic (嵌合体), consisting of intervening sequences separating the coding sequenceExons (外显子): the coding sequencesIntrons (内含子) : the intervening sequencesRNA splicing: the process by which introns are removed from the pre-mRNA.Alternative splicing (可变剪接): some pre-mRNAs can be spliced in more than one way , generating alternative mRNAs. 60% of the human genes are spliced in this manner.
102 5’splice site (5’剪接位点): the exon-intron boundary at the 5’ end of the intron Branch point site (分枝位点): an A close to the 3’ end of the intron, which is followed by a polypyrimidine tract (Py tract).
103 The intron is removed in a Form Called a Lariat (套马索) as the Flanking Exons are joined The chemistry of RNA splicingTwo successive transesterification:Step 1: The OH of the conserved A at the branch site attacks the phosphoryl group of the conserved G in the 5’ splice site. As a result, the 5’ exon is released and the 5’-end of the intron forms a three-way junction structure.
108 Exons from different RNA molecules can be fused by Trans-splicing The chemistry of RNA splicingTrans-splicing: the process in which two exons carried on different RNA molecules can be spliced together.
110 THE SPLICESOME MACHINERY RNA SplicingTopic 2THE SPLICESOME MACHINERY
111 RNA splicing is carried out by a large complex called spliceosome The spliceosome machineryRNA splicing is carried out by a large complex called spliceosomeThe above described splicing of introns from pre-mRNA are mediated by the spliceosome.The spliceosome comprises about 150 proteins and 5 snRNAs.Many functions of the spliceosome are carried out by its RNA components.
112 The five RNAs (U1, U2, U4, U5, and U6, 100-300 nt) are called small nuclear RNAs (snRNAs). The complexes of snRNA and proteins are called small nuclear ribonuclear proteins (snRNP, pronounces “snurps”).The spliceosome is the largest snRNP, and the exact makeup differs at different stages of the splicing reaction
113 Three roles of snRNPs in splicing 1. Recognizing the 5’ splice site and the branch site.2. Bringing those sites together.3. Catalyzing (or helping to catalyze) the RNA cleavage.RNA-RNA, RNA-protein and protein-protein interactions are all important during splicing.
114 RNA-RNA interactions between different snRNPs, and between snRNPs and pre-mRNA
116 Assembly, rearrangement, and catalysis within the spliceosome: the splicing pathway (Fig. 13-8) Splicing pathwaysAssembly step 11. U1 recognize 5’ splice site.2. One subunit of U2AF binds to Py tract and the other to the 3’ splice site. The former subunits interacts with BBP and helps it bind to the branch point.3. Early (E) complex is formed
117 Assembly step 21. U2 binds to the branch site, and then A complex is formed.2. The base-pairing between the U2 and the branch site is such that the branch site A is extruded(Figure 13-6). This A residue is available to react with the 5’ splice site.
121 Assembly step 4U1 leaves the complex, and U6 replaces it at the 5’ splice site.U4 is released from the complex, allowing U6 to interact with U2 (Figure 13-6c).This arrangement called the C complex.
122 B complexC complex in which the catalysis has not occurred yet
123 Catalysis Step 1:Formation of the C complex produces the active site, with U2 and U6 RNAs being brought togetherFormation of the active site juxtaposes (并置) the 5’ splice site of the pre-mRNA and the branch site, allowing the branched A residue to attack the 5’ splice site to accomplish the first transesterfication (转酯) reaction.
124 Catalysis Step 2:U5 snRNP helps to bring the two exons together, and aids the second transesterification reaction, in which the 3’-OH of the 5’ exon attacks the 3’ splice site.Final Step:Release of the mRNA product and the snRNPs
126 splicesome-mediated splicing reactions E complexA complexB complexC complex （没有该complex的图）
127 How does spliceosome find the splice sites reliably Splicing pathwaysTwo kinds of splice-site recognition errorsSplice sites can be skipped.“Pseudo” splice sites could be mistakenly recognized, particularly the 3’ splice site.
129 Reasons for the recognition errors (1) The average exon is 150 nt, and the average intron is about 3,000 nt long (some introns are near 800,000 nt)It is quite challenging for the spliceosome to identify the exons within a vast ocean of the intronic sequences.
130 (2) The splice site consensus sequence are rather loose (2) The splice site consensus sequence are rather loose. For example, only AGG tri-nucleotides is required for the 3’ splice site, and this consensus sequence occurs every 64 nt theoretically.
131 Two ways to enhance the accuracy of the splice-site selection 1. Because the C-terminal tail of the RNA polymerase II carries various splicing proteins, co-transcriptional loading of these proteins to the newly synthesized RNA ensures all the splice sites emerging from RNAP II are readily recognized, thus preventing exon skipping.
132 2. There is a mechanism to ensure that the splice sites close to exons are recognized preferentially. SR proteins bind to the ESEs (exonic splicing enhancers) present in the exons and promote the use of the nearby splice sites by recruiting the splicing machinery to those sites
133 SR proteins, bound to exonic splicing enhancers (ESEs), interact with components of splicing machinery, recruiting them to the nearby splice sites.
134 SR proteins are essential for splicing Ensure the accuracy and efficacy of constitutive splicingRegulate alternative splicingThere are many varieties of SR proteins. Some are expressed preferentially in certain cell types and control splicing in cell-type specific patterns
135 Topic 4 ALTERNATIVE SPLICING RNA SplicingTopic 4 ALTERNATIVE SPLICING
136 Single genes can produce multiple products by alternative splicing Many genes in higher eukaryotes encode RNAs that can be spliced in alternative ways to generate two or more different mRNAs and, thus, different protein products.
137 Drosophila DSCAM gene can be spliced in 38,000 alternative ways
138 There are five different ways to alternatively splice a pre-mRNA
139 Alternative splicing can be either constitutive or regulated Constitutive alternative splicing: more than one product is always made from a pre-mRNARegulative alternative splicing: different forms of mRNA are produced at different time, under different conditions, or in different cell or tissue types
140 An example of constitutive alternative splicing : Splicing of the SV40 T antigen RNA
141 Alternative splicing is regulated by activators and repressors The regulating sequences : exonic (or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and ISS). The former enhance and the latter repress splicing.Proteins that regulate splicing bind to these specific sites for their actionAlternative splicing
142 SR proteins binding to enhancers act as activators. (1) One domain is the RNA-recognition motif (RRM)(2) The other domain is RS domain rich in arginine and serine. This domain mediates interactions between the SR proteins and proteins within the splicing machinery.
143 hnRNPs binds RNA and act as repressors Most silencers are recognized by hnRNP ( heterogeneous nuclear ribonucleoprotein) family.These proteins bind RNA, but lack the RS domains. Therefore, (1) They cannot recruit the splicing machinery. (2) they block the use of the specific splice sites that they bind.
145 An example of repressors: inhibition of splicing by hnRNPI Coats the RNA and makes the exons invisible to the splicing machineryBinds at each end of the exon and conceals (隐藏) it
146 The outcome of alternative splicing: 1. Producing multiple protein products, called isoforms.2. Switching on and off the expression of a given gene. In this case, one functional protein is produced by a splicing pattern, and the non-functional proteins are resulted from other splicing patterns.
147 A small group of intron are spliced by minor spliceosome Alternative splicingThis spliceosome works on a minority of exons, and those have distinct splice-site sequence.The chemical pathway is the same as the major spliceosome.
148 U11 and U12 are in places of U1 and U2, respectively The AT-AC spliceosome
149 Topic x : Self-splicing introns RNA SplicingTopic x : Self-splicing introns自剪接内含子
150 Self-splicing introns reveal that RNA can catalyze RNA splicing Self-splicing introns: the intron itself folds into a specific conformation within the precursor RNA and catalyzes the chemistry of its own release and the exon ligationSplicing pathways
151 Adams et al., Nature 2004, Crystal structure of a self-splicing group I intron with both exons
152 Practical definition for self-splicing introns: the introns that can remove themselves from pre-RNAs in the test tube in the absence of any proteins or other RNAs.There are two classes of self-splicing introns, group I and group II self-splicing introns.
153 Three class of RNA Splicing AbundanceMechanismCatalytic MachineryNuclear pre-mRNAVery common; used for most eukaryotic genesTwo transesterification reactions; branch site AMajor spliceosomeGroup II intronsRare; some eu-Karyotic genes from organelles and prokaryotesSame as pre-mRNARNA enzyme encoded by intron (ribozyme)Group I intronsRare; nuclear rRNA in some eukaryotics, organlle genes, and a few prokaryotic genesTwo transesterific-ation reactions; exogenous GSame as group II introns
154 The chemistry of group II intron splicing and RNA intermediates produced are the same as that of the nuclear pre-mRNA.
156 Group I introns release a linear intron rather than a lariat Instead of using a branch point A, group I introns use a free G to attack the 5’ splice site.This G is attached to the 5’ end of the intron.The 3’-OH group of the 5’ exon attacks the 5’ splice site.The two-step transesterification reactions are the same as that of splicing of the group II intron and pre-mRNA introns.Splicing pathways
158 Group I introns Smaller than group II introns Share a conserved secondary structure, which includes an “internal guide sequence” base-pairing with the 5’ splice site sequence in the upstream exon.The tertiary structure contains a binding pocket that will accommodate the guanine nucleotide or nucleoside cofactor
159 The similarity of the structures of group II introns and U2-U6 snRNA complex formed to process first transesterification
162 RNA editing is another way of changing the sequence of an mRNA I. Site specific deamination :1. A specifically targeted C residue within mRNA is converted into U by the deaminase.2. The process occurs only in certain tissues or cell types and in a regulated manner.RNA editing
164 The human apolipoprotein gene Stop codeIn liverIn intestines
165 3. Adenosine deamination also occurs in cells 3. Adenosine deamination also occurs in cells. The enzyme ADAR (adenosine deaminase acting on RNA) convert A into Inosine. Insone can base-pair with C, and this change can alter the sequence of the protein.4. An ion channel expressed in mammalian brains is the target of Adenosine deamination.
166 II Guide RNA-directed uridine insertion or deletion. 1. This form of RNA editing is found in the mitochondria of trypanosomes.2. Multiple Us are inserted into specific region of mRNAs after transcription (or US may be deleted).
167 3. The addition of Us to the message changes codons and reading frames, completely altering the “meaning” of the message.4. Us are inserted into the message by guide RNAs (gRNAs) .
168 gRNAs Having three regions: anchor– directing the gRNAs to the region of mRNAs it will edit.editing region – determining where the Us will be insertedpoly-U stretch
171 Once processed, mRNA is packaged and exported from the nucleus into the cytoplasm for translation mRNA transportAll the fully processed mRNAs are transported to the cytoplasm for translation into proteins
172 Movement from the nucleus to the cytoplasm is an active and carefully regulated process. The damaged, misprocessed and liberated introns are retained in the nucleus and degraded.A typical mature mRNA carries a collection of proteins that identifies it as being ready for transport.Export takes place through the nuclear pore complex.
173 Once in the cytoplasm, some proteins are discarded and are then imported back to the nucleus for another cycle of mRNA transport. Some proteins stay on the mRNA to facilitate translation.
175 Key points Why RNA splicing is important? Chemical reaction: determination of the splice sites, the products, trans-splicingSpliceosome: splicing pathway and finding the splice sitesSelf-splicing introns and mechanismsAlternative splicing and regulation, alternative spliceosomeTwo different mechanisms of RNA editingmRNA transport-a link to translation