chapter 23 catalytic rna. 23.1 introduction 23.2 group i introns undertake self-splicing by...

Chapter 23Chapter 23

Catalytic RNA

23.1 Introduction23.2 Group I introns undertake self-splicing by transesterification23.3 Group I introns form a characteristic secondary structure23.4 Ribozymes have various catalytic activities23.5 Some introns code for proteins that sponsor mobility23.6 The catalytic activity of RNAase P is due to RNA23.7 Viroids have catalytic activity23.8 RNA editing occurs at individual bases23.9 RNA editing can be directed by guide RNAs

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

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

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

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

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 transesteri

fication

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 transesterificatio

n

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

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

Figure 23.6 Excision of the group I intron in Tetrahymena rRNA occurs by successive reactions between the occupants of the guanosine-binding site and substrate-binding site. The left exon is red, and the right exon is purple.

23.4 Ribozymes have various catalytic activities

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.


Figure 23.7 The position of the IGS in the tertiary structure changes when P1 is formed by substrate binding.


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.


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.


Figure 23.9 Catalytic reactions of the ribozyme involve transesterifications between a group in the substrate-binding site and a group in the G-binding site.


Figure 23.10 Reactions catalyzed by RNA have the same features as those catalyzed by proteins, although the rate is slower. The KM gives the concentration of substrate required for half-maximum velocity; this is an inverse measure of the affinity of the enzyme for substrate. The turnover number gives the number of substrate molecules transformed in unit time by a single catalytic site.


Figure 23.11 An intron codes for an endonuclease that makes a double-strand break in DNA. The sequence of the intron is duplicated and then inserted at the break.

23.5 Some introns code for proteins that sponsor mobility

Figure 16.18 Retrotransposition of non-LTR elements occurs by nicking the target to provide a primer for cDNA synthesis on an RNA template.

23.5 Some introns code for proteins that sponsor mobility

Figure 23.12 Reverse transcriptase coded by an intron allows a copy of the RNA to be inserted at a target site generated by a double-strand break.

23.5 Some introns code for proteins that sponso

r mobility

Viroid is a small infectious nucleic acid that does not have a protein coat.

23.6 RNA can have ribonuclease activities

Figure 23.10 Reactions catalyzed by RNA have the same features as those catalyzed by proteins, although the rate is slower. The KM gives the concentration of substrate required for half-maximum velocity; this is an inverse measure of the affinity of the enzyme for substrate. The turnover number gives the number of substrate molecules transformed in unit time by a single catalytic site.


Figure 12.16 The rolling circle generates a multimeric single-stranded tail.


Figure 23.13 Self-cleavage sites of viroids and virusoids have a consensus sequence and form a hammerhead secondary structure by intramolecular pairing. Hammerheads can also be generated by pairing between a substrate strand and an "enzyme" strand.


Figure 23.14 A hammerhead ribozyme forms a V-shaped tertiary structure in which stem 2 is stacked upon stem 3. The catalytic center lies between stem 2/3 and stem 1. It contains a magnesium ion that initiates the hydrolytic reaction.


Figure 23.15 The sequence of the apo-B gene is the same in intestine and liver, but the sequence of the mRNA is modified by a base change that creates a termination codon in intestine.

23.7 RNA editing utilizes information fro

m several sources

Figure 23.16 Editing of mRNA occurs when a deaminase acts on an adenine in an imperfectly paired RNA duplex region.

23.7 RNA editing utilizes information from several sources

Figure 23.17 The mRNA for the trypanosome coxII gene has a -1 frameshift relative to the DNA; the correct reading frame is created by the insertion of 4 uridines.


Figure 23.18 Part of the mRNA sequence of T. brucei coxIII shows many uridines that are not coded in the DNA (shown in red) or that are removed from the RNA (shown as T).


Figure 23.19 Pre-edited RNA base pairs with a guide RNA on both sides of the region to be edited. The guide RNA provides a template for the insertion of uridines. The mRNA produced by the insertions is complementary to the guide RNA.


Figure 23.20 The Leishmania genome contains genes coding for pre-edited RNAs interspersed with units that code for the guide RNAs required to generate the correct mRNA sequences. Some genes have multiple guide RNAs.


Figure 23.21 Addition or deletion of U residues occurs by cleavage of the RNA, removal or addition of the U, and ligation of the ends. The reactions are catalyzed by a complex of enzymes under the direction of guide RNA.

23.7 RNA editing utilizes information fro

m several sources

23.8 Summary

chapter 23 catalytic rna. 23.1 introduction 23.2 group i introns undertake self-splicing by...

Documents

rna splicing

transesterification

catalytic rna slide

introduction slide

introns code

successful splicing

rna editing

linear rna