分子生物学Chapter 9 RNA Processing.docx

1、分子生物学Chapter 9 RNA ProcessingChapter 9 RNA Processing IntroductionVery few RNA molecules are transcribed directly into the final mature RNA.v Most newly transcribed RNA molecules (primary transcripts) undergo various alterations to yield the mature productv RNA processing is the collective term used

2、 to describe the molecular events allowing the primary transcripts to become the mature RNA. 1.Basic Features of RNA Processing v Prokaryotic RNA2-5 half life; polycistronic; seldom modified; used immediatelyCoupled transcription and translation; many ribosomes bind same operon, and synthesize polyp

3、eptide simultaneously . called polyribosomes or polysomes for short.Polycistronic mRNAs: coordinate expression of 1 gene: operonMultiple translational start sites per mRNAEukaryotic RNA: v long half life; monocistronic; always modified; transport to cytoplasmv Most post-transcription modifications o

4、f the primary transcript occur in nucleus, before transport to cytoplasm.v these modifications convert the transcript (Heterogeneous Nuclear RNA hnRNA) into mRNA1.1 Major Eukaryotic Modifications events v G capping of 5 end of pre-mRNACleavage and Poly(A) addition to 3 endSplicing out (removal of) I

5、ntronsSome methylation of bases1.1.1. Poly(A) on 3 endv Transcripts are cleaved at 3 end past a highly conserved AAUAAA sequence.Such Cleavage involves some protein cofactors v The Poly(A) polymerase (PAP) adds 200 A residues to most mRNAsv Poly(A)-Binding Protein (PABP) binds to the poly(A) stoichi

6、ometrically, one protein every 10-20 bases . increases stability and enhances translationv Histone mRNAs are poly(A)-free.1.1.2 Methylated G Cap at 5 end: v Guanylyl transferase catalyzes addition of GTP in 5-5 direction, with release of PPi from the GTP, and release of Pi from the terminal triphosp

7、hate of the RNA transcript.Then one of 3 methyl Capping reactions occurs v A) Cap 0: a methyl group is added to the G-7 posn: Guanine-7-MethylTransferasePresent in all eukaryotic mRNA . no further capping in unicelluar eukaryotesv B) Cap 1: a methyl group is then added to the 2-OH of the ribose of t

8、he 1st nucleotide in the original transcript: 2-O-MethylTransferasePresent in most eukaryotic mRNA . if base is A, it too can be methylatedv C) Cap 2: a similar methylation of the 2nd nucleotide of the original transcriptPresent in about 10-15% of total capped population. Cap at 5 end1.1.3. Internal

9、 Methylation:v N-6 methylation of Adenines, at frequency of about 0.1%1.2. Eukaryotic RNA Splicing: Introns and Exons v Eukaryotic genes are often interrupted genes:Coding sequence is interrupted by noncoding sequences .v Removal of the Introns in RNA transcript modification is called RNA splicing v

10、 Exons are Expressed sequences: these sequences are those present in mature mRNAv Introns are InterVening Sequences (IVS), sequences found between Exon sequences in the genomic DNA, i.e. in the gene Chapter 9 RNA Processing 2. Nuclear RNA splicing v RNA splicing occurs in the nucleus, on the initial

11、 transcripts (pre-mRNA or hnRNA) complexed with the nuclear proteins: hnRNP particles.2.1. GU-AG rulev GU always at 5 end of intron: Donor site: AG | GUAAGUAG always at 3 end of intron: Acceptor site: (Py.Py) 12 NCAG | N2.2. Spliceosomes v Spliceosomes are composed of Splicing Factor proteins (40) p

12、lus small nuclear RNAs (snRNA) found in nucleoprotein (10 proteins) complexes . 50-60 S size .These ribonucleoprotein complexes are called snRNP particles, or snurpssnRNAs.v The snRNPs are U1, U2, U5, and U4/U6, containing U1, U2, U4, U5, U6 snRNAs. Functional roles for each snRNP:snRNP U1 :v v Stru

13、cture: 4 stem-loop Domains (A,B,C,D)Sm-binding site: non-paired region between Domains C and B - binding of snRNP proteins5 non-paired end: H-bonds to Donor site of pre-mRNA . This pairing is essential for splicing; some pairing sites more important than othersv U1 -initially bound to 5-splice sitev

14、 -is released upon recruitment of U4/U5/U6 v (determines which 5-splice site is used?)U1 SnRNAv Mutations that abolish function of the 5splicing site can be suppressed by compensating mutations in snRNA that restore base pairingU2 snRNAv -initially binds the branchpoint recognition sequence v -forms

15、 two duplexes with U6, bringing the intron 5splice site close to the branchpoin U4 RNAv -initially complexed with U5 and U6v -keeps U6 in an unfolded conformationv -is released after delivering U6 to the 5-splice sitev (U6 chaperone?)U5 RNAv -initially complexed with U4 and U6v -binds exon sequences

16、 upstream of the 5-splice sitev and downstream of the 3-splice sitev (guide sequence for holding exons together?)U6 RNAv -initially complexed with U4 and U5v -displaces U1 from 5-splice site, forming duplex with intron sequencesv -complexes with U2, bringing the intron 5-splice site close to the bra

17、nchpoint.v (catalytic activity?)v U6-U4 pairing is incompatible with U6-U2 pairing. When U6 joins the spliceosome it is paired with U4. Release of U4 allows a conformational change in U6; one part of the released sequence forms a hairpin, and the other part pairs with U2. Because an adjacent region

18、of U2 is already paired with the branch site, this brings U6 into juxtaposition with the branch.2.3 Splicing Reaction: v Stage 1: Donor Site is cut, 5 end is covalently attached to a Branch site in the Intron in a 5-2 phosphodiester bond forming a Lariat Intermediate .v Stage 2: Acceptor Site is cut

19、, exons are joined, intron is released as the Lariatv Stage 3: The Lariat is debranched and degraded.Splicing of exons in pre-mRNA occurs via two transesterification reactions v OH- nucleophilic attack on 5-P of intron: 1st transesterification reactionv Exon A 3-OH nucleophilic attack on 3-P of intr

20、on: 2nd transesterification reactionv Exons joined; Intron excised, either as linear RNA or as branched lariat structure2.4 Role of snRNP particles in the 3 Stages(1) v 1). U1 snRNP binds to Donor site .v 2) U2 snRNP H-bonds to Branch site of Intron, using ATP hydrolysis and involving a snRNP auxill

21、iary factor (U2AF): Commitment to Splicing.v 3) U1 snRNP interacts with U2 snRNP, bringing Donor site and Branch site together . Role of snRNP particles in the 3 Stages(2)v 4) U5/U4/U6 trimer complex now binds to U1 and U2, with ATP hydrolysis; U5 binds the intron Donor Site; U6 binds U2: This is th

22、e complete Spliceosome. ATP provides energy in each of these reactions for conformational changes . U4 interacts with U6 via H-bonds.2.4 Role of snRNP particles in the 3 Stages(3)v 5) U5 shifts position from the intron part to the exon part of Donor site, and U2 H-bonds with U6, displacing U4 from i

23、ts interaction with U6 1st Transesterification reaction now occurs; enzyme may be the U2-U6 complexv 6) U5 snRNP now interacts with the intron Acceptor site, as well as with U2, catalyzing cutting at the intron-exon junction and joining of the two Exons 2nd Transesterification Reaction2.4 Role of sn

24、RNP particles in the 3 Stages(4)v 7) Spliceosome is now disassembled, with release of U1, U2, U4, U5, U6, joined exons, and intron Lariat v 8) Intron Lariat is Debranched and degraded The RNA splicing mechanism. v The splicing reaction proceeds through discrete stages in which spliceosome formation

25、involves the interaction of components that recognize the consensus sequences. Chapter 9 RNA Processing 3. Group II Introns v fungal mitochondrial introns . fairly uncommon.v Similar to nuclear introns: GU-AG rule; splicing via a Lariat intermediate; the two transesterification reactions are similar

26、: . BUT . Intron is Self-Splicing. v v Three classes of splicing reactions proceed by two transesterifications.Schematic diagrams comparing the secondary structures of group II self-splicing introns (a) and U snRNAs present in the spliceosome (b). v Branch site A-OH - Donor site G; Donor site G-OH -

27、 Acceptor site Av Domains 5 and 6 in RNA 2 structure are similar to U2-U6 and U2-intron interactions; this provides the active site for the RNA catalytic activity.v ：v Nuclear splicing and group II splicing involve the formation of similar secondary structures. The sequences are more specific in nuc

28、lear splicing; group II splicing uses positions that may be occupied by either purine (R) or either pyrimidine (Y). Chapter 9 RNA Processing 4.rRNA Processing v Group I Introns v Found in rRNA genes of lower eukaryotes and in fungal mitochondrial genesAlso found in three genes in E. coli phage T4 !

29、. the only example of prokaryotic introns.v Two common properties:1. Self-Splicing: ability of the RNA to splice itself . intramolecular . RNA catalyst2. Characteristic Secondary Structure of 9 stem-loops Splicing mechanisms in group I and group II self-splicing introns and spliceosome-catalyzed spl

30、icing of pre-mRNA 4.1. Tetrahymena rRNA v Precursor: 35S processed, as above, into - 18S, 5.8S, 28S rRNA28S has one 400 base Intron in some Tetrahymena strains:This Intron can Self-Splice; requires ions and a G-nuc (GTP, GDP, GMP, GR) 4.2. Self-Splicing Reaction1) G 3-OH attacks 5 end of Intron2) 3-

31、OH of Exon A attacks 5 end of Exon B: exons join3) Linear Intron, with G(414) at 5 end, now circularizes 2) 4.3 Circularization of the Linear Intron:v 1) 3-OH of Intron, on G(414) , DIFFERENT from the the free G above, attacks pA(16) or pU(20) , yielding cyclized Intron and splitting off 5end 15 or 19 nucs from the Intronv 2) Water hydrolyzes the cyclized Intron at SAME positionv 3) If First cyclization were at pA(16), then 3-OH of Intron attacks pU(20) , yielding cyclized Intron and sp