mol gen 7CatalyticRNA

Catalytic RNA

Lecture Information

  • Instructor: Dr. Frank Stearns

  • Course: BIO 330

  • Term: Spring 2025

Lecture Objectives

  • Describe the mechanisms of Group I and Group II intron self-splicing

  • Understand how ribozymes act as enzymes

  • Explain the catalytic activity of RNase P

  • Compare and contrast viroids and virisoids, and explain their catalytic activities

  • Describe RNA editing mechanisms

  • Differentiate between RNA splicing and protein splicing

21.1 Introduction

Definition of Ribozyme

  • Ribozyme: An RNA molecule with catalytic activity.

  • In ribosomes, the RNA component is catalytic while proteins provide scaffolding.

  • Ribozyme actions can be either intermolecular or intramolecular.

  • Typically involve cleavage or joining of phosphodiester bonds.

RNA Editing

  • RNA editing: Changes to RNA sequence after transcription.

21.2 Group I Introns Undertake Self-Splicing by Transesterification

Requirements for Self-Splicing

  • In vitro Conditions: Requires two metal ions and a guanosine nucleotide (GTP, GDP, GMP, or guanosine).

  • Energy: No external energy required.

Mechanism of Self-Splicing

  • Two transesterification reactions facilitate splicing without energy input.

Reaction Process

  1. 3′–OH end of guanosine attacks the 5′ end of the intron during the first transesterification.

  2. The newly formed 3′–OH end attacks the junction between intron and second exon in the second transesterification.

  3. The intron exits as a linear structure, which can circularize through an internal base pair interaction.

21.3 Group I Introns Form a Characteristic Secondary Structure

Structural Features

  • Composed of nine duplex regions forming a secondary structure.

  • Catalytic activity mainly localized in regions P3, P4, P6, and P7.

Specific Pairing

  • Both regions P4 and P7 incorporate conserved consensus sequences.

  • A sequence adjacent to P7 base pairs with the reactive guanosine.

21.4 Ribozymes Have Various Catalytic Activities

Customizing Ribozymes

  • Modification of substrate binding sites in group I introns can allow interaction with different sequences associated with reactive G.

  • Capable of both splicing and RNA ligase activities.

Kinetics and Theoretical Basis

  • Ribozymes exhibit classical enzyme kinetics, though with lower catalytic rates.

  • Proposed that 2′–OH bond reactions may have led to early catalytic activities in RNA.

Riboswitches and Synthetic Constructs

  • Riboswitch: A catalytic RNA responding to specific small ligands, influencing transcription and translation.

  • Novel synthetic RNA with RNA polymerase activity has been developed, supporting the RNA world hypothesis.

21.5 Some Group I Introns Encode Endonucleases That Sponsor Mobility

Mechanism of Mobility

  • Mobile introns can insert themselves into new genomic sites through specific endonuclease activity following a double-strand break.

Intron Homing

  • Process where certain introns insert into target DNA without targeting sites needing homology.

  • Highly specific reactions focus on a precise target sequence.

21.6 Group II Introns May Encode Multifunction Proteins

Autosplicing Requirements

  • Group II introns can self-splice but often need protein assistance encoded in the intron.

  • One reading frame encodes multifunctional protein activities including reverse transcriptase and DNA endonuclease.

Functionality

  • The endonuclease cleaves DNA for intron insertion, while reverse transcriptase synthesizes DNA from RNA.

21.7 Some Autosplicing Introns Require Maturases

Catalytic Assistance

  • Certain autosplicing introns need maturase functions to fold into the active structure necessary for catalysis.

21.8 The Catalytic Activity of RNase P Is Due to RNA

Overview of RNase P

  • RNase P: Ribonucleoprotein complex where the RNA component exhibits catalytic activity.

  • Crucial for functionality across bacteria, archaea, and eukaryotic cells.

  • Related ribonuclease MRP in eukaryotes contributes to rRNA processing and destruction of cyclin B mRNA.

21.9 Viroids Have Catalytic Activity

Characteristics of Viroids and Virusoids

  • Viroids possess a hammerhead structure enabling self-cleavage; deemed small RNA plant pathogens.

  • Differences: Viroids are non-encapsulated, whereas virusoids are encapsulated within plant viruses.

Mechanism of Cleavage

  • Hammerhead structures typically contain conserved regions that guide catalytic activity, able to cleave complementary substrate strands.

21.10 RNA Editing Occurs at Individual Bases

Specific Editing Examples

  • Example interactions: Deaminations in apolipoprotein-B and glutamate receptor mRNAs change their coding sequences post-transcription.

21.11 RNA Editing Can Be Directed by Guide RNAs

Guide RNA Functions

  • In trypanosomes, RNA editing involves uridine insertions and deletions, guided by specific RNA templates.

  • The editosome complex orchestrates editing through various enzymatic activities to add or remove uridines.

21.12 Protein Splicing Is Autocatalytic

Inteins and Exteins

  • Inteins catalyze their own excision from larger proteins, merging the surrounding exteins together.

  • Many inteins possess dual functionality, such as serving as an endonuclease while performing protein splicing.