清华大学：《分子生物学》课程PPT教学课件（基因ene）第二十三章 催化RNA（Catalytic RNA）

Chapter 23 Catalytic RNA 清革大当

Chapter 23 Catalytic RNA

23.1 Introduction 23.2 Group I introns undertake self-splicing by transesterification 23.3 Group I introns form a characteristic secondary structure 23.4 Ribozymes have various catalytic activities 23.5 Some introns code for proteins that sponsor mobility 23.6 The catalytic activity of RNAase P is due to RNA 23.7 Viroids have catalytic activity 23.8 RNA editing occurs at individual bases 23.9 RNA editing can be directed by guide RNAs 清莘大当

23.1 Introduction 23.2 Group I introns undertake self-splicing by transesterification 23.3 Group I introns form a characteristic secondary structure 23.4 Ribozymes have various catalytic activities 23.5 Some introns code for proteins that sponsor mobility 23.6 The catalytic activity of RNAase P is due to RNA 23.7 Viroids have catalytic activity 23.8 RNA editing occurs at individual bases 23.9 RNA editing can be directed by guide RNAs

23.1 Introduction The idea that only proteins have enzymatic activity was deeply rooted in biochemistry. The enzyme ribonuclease P is a ribonucleoprotein that contains a single RNA molecule bound to a protein. Small RNAs of the virusoid class have the ability to perform a self-cleavage reaction. Introns of the group I and group II classes possess the ability to splice themselves out of the pre-mRNA that contains them. 清菜大兰

The idea that only proteins have enzymatic activity was deeply rooted in biochemistry. The enzyme ribonuclease P is a ribonucleoprotein that contains a single RNA molecule bound to a protein. Small RNAs of the virusoid class have the ability to perform a self-cleavage reaction. Introns of the group I and group II classes possess the ability to splice themselves out of the pre-mRNA that contains them. 23.1 Introduction

23.1 Introduction The common theme of these reactions is that the RNA can perform an intramolecular or intermolecular reaction that involves cleavage or joining of phosphodiester bonds in vitro. RNA splicing is not the only means by which changes can be introduced in the informational content of RNA. 情菜大当

The common theme of these reactions is that the RNA can perform an intramolecular or intermolecular reaction that involves cleavage or joining of phosphodiester bonds in vitro. RNA splicing is not the only means by which changes can be introduced in the informational content of RNA. 23.1 Introduction

23.2 Group I introns undertake self- splicing by transesterification Exon 1 Intron Exon 2 N八N入入八入NN Figure 23.1 Splicing of the Tetrahymena 35S rRNA precursor Gel electrophoresis Transcripti can be followed by gel 35S RNA electrophoresis.The removal of the intron is revealed by the Splicing appearance of a rapidly moving small band.When the intron becomes circular,it Cyclization electrophoreses more slowly,as seen by a higher band Circular intron Unear intron 清苇大当

Figure 23.1 Splicing of the Tetrahymena 35S rRNA precursor can be followed by gel electrophoresis. The removal of the intron is revealed by the appearance of a rapidly moving small band. When the intron becomes circular, it electrophoreses more slowly, as seen by a higher band. 23.2 Group I introns undertake self￾splicing by transesterification

23.2 Group I introns undertake self- splicing by transesterification fa过transter 多9的809 ttacks G-OH Figure 23.2 Self-splicing 53 5 Exon B3 pG-OH pN pNpNpN pN pNp以单XXp式p occurs by oNpNpNpNpNpN-OH transesterification pGpXpXpXpXpXpx reactions in which bonds Second transfer are exchanged directly. 8888m合aca The bonds that have been generated at each stage are indicated by Third transfer 983658a弯瓶 the shaded boxes. 情華大兰

Figure 23.2 Self-splicing occurs by transesterification reactions in which bonds are exchanged directly. The bonds that have been generated at each stage are indicated by the shaded boxes. 23.2 Group I introns undertake self￾splicing by transesterification

3-OH of G attacks pAor pu SG.UUUPACCUPUUG 5G.UUUpACCUpUUG 23.2 Group I 414 introns undertake Cyclization self-splicing by UG transesterification Reverse cyclization Figure 23.3 The excised intron can form circles by Secondary cyclization using either of two internal OH Linearization OH sites for reaction with the 5 414 end,and can reopen the L15 RNA L19 RNA trans reaction circles by reaction with 16 20 UUUPACCUPUUG water or oligonucleotides. 清菜大当

Figure 23.3 The excised intron can form circles by using either of two internal sites for reaction with the 5 end, and can reopen the circles by reaction with water or oligonucleotides. 23.2 Group I introns undertake self-splicing by transesterification

23.2 Group I introns -OH Cs pairs with undertake self- 595 IGS site near 5' end of RNA splicing by transesterification G-OH attacks CC-OH 3 CpC bond GGAGE-5 Figure 23.8 The L-19 linear HO-CCCCn RNA can bind C in the C is transferred to substrate-binding site;the C-OH 3'-G;C4IS GGGAGE-5 released reactive G-OH 3 end is located in the G-binding site, C-OH and catalyzes transfer reactions Another Cs binds CC-OH 3 transfer reaction is reversed that convert 2 C5 HO-CCCCCCp oligonucleotides into a C4 and C6 is released, a C6 oligonucleotide eraing.19 情菜大兰

Figure 23.8 The L-19 linear RNA can bind C in the substrate-binding site; the reactive G-OH 3 end is located in the G-binding site, and catalyzes transfer reactions that convert 2 C5 oligonucleotides into a C4 and a C6 oligonucleotide. 23.2 Group I introns undertake self￾splicing by transesterification

23.3 Group I introns form a characteristic secondary structure Exor G-OH P Figure 23.4 Group I introns have a 5'cUcUCU First 5'UGCGGGB 3'GGGAGG transfer 3'ACGCCC common secondary structure that is IGS Q formed by 9 base paired regions. The sequences of regions P4 and P7 are conserved,and identify the P P3 P6 individual sequence elements P,Q. Exon P7 R,and S.PI is created by pairing Exon 2 between the end of the left exon P8 and the IGS of the intron;a region between P7 and P9 pairs with the 3 X0X8 2 bp fomm at 3'end end of the intron. R of intron 清苇大当

Figure 23.4 Group I introns have a common secondary structure that is formed by 9 base paired regions. The sequences of regions P4 and P7 are conserved, and identify the individual sequence elements P, Q, R, and S. P1 is created by pairing between the end of the left exon and the IGS of the intron; a region between P7 and P9 pairs with the 3' end of the intron. 23.3 Group I introns form a characteristic secondary structure

23.3 Group I introns form a characteristic secondary structure Figure 23.5 Placing the ntron inserted in codon 10 Promoter Tetrahymena intron within the AUG B-galactosidase codon codons Intron nosidase b-galactosidase coding sequence creates an assay for self-splicing 门 in E.coli.Synthesis of b- Transcription galactosidase can be tested by Splicing adding a compound that is turned blue by the enzyme.The sequence is carried by a B-galactosidase bacteriophage,so the presence of blue plaques indicates Blue plaques generated 00o successful splicing. by staining for B- galactosidase 清菜大当

Figure 23.5 Placing the Tetrahymena intron within the b-galactosidase coding sequence creates an assay for self-splicing in E. coli. Synthesis of b￾galactosidase can be tested by adding a compound that is turned blue by the enzyme. The sequence is carried by a bacteriophage, so the presence of blue plaques indicates successful splicing. 23.3 Group I introns form a characteristic secondary structure