Catalytic RNA Vocabulary

Catalytic RNA

Lecture Objectives

  • Describe the mechanisms of Group I and Group II intron self-splicing.
  • Describe how ribozymes act as enzymes.
  • Explain the catalytic activity of RNase P.
  • Compare and contrast viroids and virusoids and explain their catalytic activity.
  • Describe the mechanisms of RNA editing.
  • Compare and contrast RNA splicing and protein splicing.

21.1 Introduction

  • Ribozyme: An RNA molecule with catalytic activity.
  • In ribosomes, RNA is catalytic, while protein provides scaffolding.
  • Ribozymes can act either inter- or intramolecularly.
  • They often (but not always) involve the cleavage or joining of phosphodiester bonds.
  • RNA editing: A change of sequence at the RNA level following transcription.

21.2 Group I Introns Undertake Self-Splicing by Transesterification

  • The only factors required for autosplicing (or self-splicing) in vitro by group I introns are two metal ions and a guanosine nucleotide.
  • May be GTP, GDP, GMP, or just guanosine – no energy needed.
  • Splicing occurs by two transesterification reactions, without requiring input of energy.
  • The 3’–OH end of the guanosine cofactor attacks the 5’ end of the intron in the first transesterification.
  • The 3’–OH end generated at the end of the first exon attacks the junction between the intron and second exon in the second transesterification.
  • The intron is released as a linear molecule that circularizes when its 3’–OH terminus attacks a bond at one of two internal positions.

21.3 Group I Introns Form a Characteristic Secondary Structure

  • Group I introns form a secondary structure with nine duplex regions.
  • The cores of regions P3, P4, P6, and P7 have catalytic activity.
  • Regions P4 and P7 are both formed by pairing between conserved consensus sequences.
  • A sequence adjacent to P7 base pairs with the sequence that contains the reactive G.

21.4 Ribozymes Have Various Catalytic Activities

  • By changing the substrate binding site of a group I intron, it is possible to introduce alternative sequences that interact with the reactive G.
  • Can have splicing or RNA ligase activity.
  • The reactions follow classical enzyme kinetics with a low catalytic rate.
  • Reactions using 2’–OH bonds could have been the basis for evolving the original catalytic activities in RNA.
  • Riboswitch: A catalytic RNA whose activity responds to a small ligand.
    • Alters transcriptional attenuation or translational initiation.
  • Synthetic RNA constructs that have RNA polymerase activity have been constructed.
  • RNA can direct its own synthesis.
  • Support RNA world.

21.5 Some Group I Introns Encode Endonucleases That Sponsor Mobility

  • Mobile introns are able to insert themselves into new sites.
  • Mobile group I introns encode an endonuclease that makes a double-strand break at a target site.
  • The intron transposes into the site of the double-strand break by a DNA-mediated replicative mechanism.
  • Intron homing: The ability of certain introns to insert themselves into a target DNA.
  • No homology among target sites.
  • Targets are among the longest for endonucleases.
  • The reaction is therefore very specific for a single target sequence.

21.6 Group II Introns May Encode Multifunction Proteins

  • Group II introns can autosplice in vitro but are usually assisted by protein activities encoded in the intron.
  • A single reading frame specifies a protein with reverse transcriptase activity, maturase activity, a DNA-binding motif, and a DNA endonuclease.
  • The endonuclease cleaves target DNA to allow insertion of the intron at a new site.
  • The reverse transcriptase generates a DNA copy of the inserted RNA intron sequence.

21.7 Some Autosplicing Introns Require Maturases

  • Autosplicing introns may require maturase activities encoded within the intron to assist folding into the active catalytic structure.

21.8 The Catalytic Activity of RNase P Is Due to RNA

  • Ribonuclease P (RNase P) is a ribonucleoprotein in which the RNA has catalytic activity.
  • RNase P is essential for bacteria, archaea, and eukaryotes.
  • RNase MRP in eukaryotes is related to RNase P and is involved in rRNA processing and degradation of cyclin B mRNA.

21.9 Viroids Have Catalytic Activity

  • Viroids and virusoids form a hammerhead structure that has a self-cleaving activity.
  • Small plant RNA pathogens similar to viruses.
    • Viroids: not encapsulated.
    • Virusoids: encapsulated in plant viruses.
  • Similar structures can be generated by pairing a substrate strand that is cleaved by an enzyme strand.
  • When an enzyme strand is introduced into a cell, it can pair with a substrate strand target that is then cleaved.
  • Consensus hammerheads have three stem loops and conserved bases GNCG, CNGAGN, AA.

21.10 RNA Editing Occurs at Individual Bases

  • Apolipoprotein-B and glutamate receptor mRNAs have site-specific deaminations catalyzed by cytidine and adenosine deaminases that change the coding sequence.

21.11 RNA Editing Can Be Directed by Guide RNAs

  • Extensive RNA editing in trypanosome mitochondria occurs by insertions or deletions of uridine.
  • The substrate RNA base pairs with a guide RNA on both sides of the region to be edited.
  • The guide RNA provides the template for addition (or less often, deletion) of uridines.
  • Editing is catalyzed by the editosome, a complex of endonuclease, exonuclease, terminal uridyl transferase activity, and RNA ligase.

21.12 Protein Splicing Is Autocatalytic

  • An intein has the ability to catalyze its own removal from a protein in such a way that the flanking exteins are connected.
  • Protein splicing is catalyzed by the intein.
  • Most inteins have two independent activities: protein splicing and a homing endonuclease.