分子生物学chapter 5.ppt

Chapter 5 mRNA Modifications in Eukaryotes 第5章 真核生物mRNA的 修饰,,,In prokaryotes, transcription produces a nearly exact mRNA copy of the DNA, and the transcript is immediately translated into protein. In eukaryotes, a series of modifications occur to mRNA during and after transcription.,在原核生物中，转录产生的mRNA几乎是DNA的准确拷贝，并且这一转录产物会立即被转译成蛋白质在真核生物中，转录时以及转录后会对mRNA进行一系列修饰mRNA Modifications in Eukaryotes,,,5.1 Capping 5.2 Polyadenylation 5.3 Splicing 5.4 mRNA Editing 5.5 Experiments,5.1 加帽 5.2 聚腺苷酸化 5.3 剪接 5.4 mRNA编辑 5.5 实验研究,,,Chapter 5 mRNA Modifications in Eukaryotes,concept,The remove of nucleotides by both endonucleases and exonucleases. The addtion of nucleotides to the 5’-or 3’-ends of the primary transcription or their cleavage product.,A primary transcript or pre-mRNA is an mRNA that has been transcribed but is not yet ready for translation.,RNA processing is the collective term used to describe these alterations to the primary transcript.,,5.1 Capping / 加帽,Capping is the process of adding a derivative(m7G) of guanine nucleotide to the 5’ end of the pre-mRNA.,This derivative is attached to the 5’ end of the pre-mRNA by a 5’-5’ triphosphate bond linkage. The reaction is carried out by an enzyme called guanyltransferase(鸟苷转移酶).,,Structure of the cap / 帽的结构,,5’-3’ phospho- diester bond,,5’-5’ triphosphate bond,,7-methylguanine nucleotide,methyl group,Capping takes place quite early.,Transcription of an mRNA occurs in the 5’ to 3’ direction. So, the 5’ end of the mRNA comes out first. Capping takes place quite early, before the rest of the gene is transcribed.,Capping process,Inverted guanine nucleotide,CTD: C-terminal domain,These three enzymes stay together around the C-terminal domain of the Rpb2 subunit of RNA polymerase II. They act together to complete the capping process.,,,Functions of the cap structure,1. Helps prevent degradation 帮助防止降解 2. Helps transport into cytoplasm 帮助转运到细胞质中 3. Enhances translation / 增强转译 4. Helps remove the first intron 帮助去除第一个内含子,1. Helps prevent degradation,RNAs in the cell can be rapidly degraded by ribonucleases. However, these enzymes are generally not capable of degrading the triphosphate bond in the cap structure.,,,2. Helps transport into cytoplasm,Cap structure helps the RNA transcript to pass through selective pores of the nuclear membrane and into the cytoplasm.,3. Enhances translation / 增强转译,In order to bind the ribosome, mRNA requires help from cap-binding protein. this protein requires the presence of the cap structure.,ribosome,4. Helps remove the first intron,Cap is required for mRNA splicing, to occur completly. Specifically, its presence is required for splicing of the first intron.,AAAAAAA - - - - - - AAAA,5.2 Polyadenylation / 聚腺苷酸化,,,Poly(A) tail,,,,,Polyadenylation,Pre-mRNA,Polyadenylation is a modification process in which a string of about 250 adenine nucleotides is added to the 3’ end of the transcript.,,,,,Polyadenylation signal / 聚腺苷酸化信号,AAAAAAA - - - - - - AAAA,,,Poly(A) tail,,,,,Polyadenylation,Pre-mRNA,Polyadenylation signal has a typical typical sequence of AAUAAA. This sequence gives an instruction as “to cut the mRNA about 20 nucleotides downstream, near a GU-rich sequence”.,,AAUAAA,GU,,AAUAAA motif by itself not sufficient for polyadenylation.,If it were, then polyadenylation would occur downstream of the many AAUAAA sequence found in introns, but it does not.,Polyadenylation Signals:,50-250 poly(A),Cleavage and polyadenylation of a pre-mRNA,polyadenylation involves both cleavage of the pre-RNA and polyadenylation at the cleavage site.,Requires several proteins: CPSF （切割与聚腺苷酸化特异因子）, CstF （切割激活因子）, CFI , CFII （切割因子I和II）, poly(A)polymerase, and RNA polymerase II(in particular , the CTD of RPb2).,The cleavage complex / 切割复合体,Cleavage complex,CPSF (cleavage and polyadenylation specificity factor),CstF (cleavage stimulation factor),CFI (cleavage factor I),CFII (cleavage factors II),,,,,AAUAAA signal has been transcribed, CPSF binds to the AAUAAA sequence.,CstF binds to the G-U rich sequence,PBP can help poly(A) polymerase to work more efficiently.,CTD: C-terminal domain,,,Proteins for capping at CTD,Proteins for tailing at CTD,,after capping is performed, CTD will be phosphorylated Gradually and the capping proteins are removed.,,,Functions of the poly(A) tail poly(A)尾的功能,,,,,,,,,,,,,,AAAAAAA - - - - - - AAAA,,,,,,,,AAAAAAA - - -,A,A,A,A,A,A,,,The main function of poly(A) tail is to protect the mRNA from degradation by ribonucleases.,It depends on the long string of As that separate the coding region from the end of the mRNA,,,AAAAAAA - - - - - - AAAA,There is also some evidence that the poly-A tail is involved in splicing and enhances translation of mRNAs.,,,5.3 Splicing / 剪接,,,Exons: Parts of a gene that are expressed as protein.,Introns: Sequences that do not code for protein and interrupt the coding regions.,,Splicing: The process of removing introns and rejoin the exon from a pre-mRNA.,Interrupt gene,,Junction sequence of intron linking wit。