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    RNA Catalysis

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    PREETI MANGAL
    This document summarizes RNA catalysis. It discusses naturally occurring RNA catalysts like ribozymes that catalyze reactions like RNA cleavage, splicing, and peptide bond formation. It provides examples of small self-cleaving ribozymes like the hammerhead, hairpin, VS, and HDV ribozymes. Larger RNA catalysts are discussed like RNase P and group I and II introns. Crystal structures of ribozymes like the hammerhead, hairpin, HDV, and group I introns are described. The peptidyl transferase activity of the ribosome is noted, with its crystal structure showing RNA is responsible for catalysis. In vitro selection has identified many nucleic acid catalysts for diverse

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    RNA Catalysis

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    PREETI MANGAL
    23 views37 pages
    This document summarizes RNA catalysis. It discusses naturally occurring RNA catalysts like ribozymes that catalyze reactions like RNA cleavage, splicing, and peptide bond formation. It provides examples of small self-cleaving ribozymes like the hammerhead, hairpin, VS, and HDV ribozymes. Larger RNA catalysts are discussed like RNase P and group I and II introns. Crystal structures of ribozymes like the hammerhead, hairpin, HDV, and group I introns are described. The peptidyl transferase activity of the ribosome is noted, with its crystal structure showing RNA is responsible for catalysis. In vitro selection has identified many nucleic acid catalysts for diverse

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    This document summarizes RNA catalysis. It discusses naturally occurring RNA catalysts like ribozymes that catalyze reactions like RNA cleavage, splicing, and peptide bond formation. It provides examples of small self-cleaving ribozymes like the hammerhead, hairpin, VS, and HDV ribozymes. Larger RNA catalysts are discussed like RNase P and group I and II introns. Crystal structures of ribozymes like the hammerhead, hairpin, HDV, and group I introns are described. The peptidyl transferase activity of the ribosome is noted, with its crystal structure showing RNA is responsible for catalysis. In vitro selection has identified many nucleic acid catalysts for diverse

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    23 views37 pages

    RNA Catalysis

    Uploaded by

    PREETI MANGAL
    This document summarizes RNA catalysis. It discusses naturally occurring RNA catalysts like ribozymes that catalyze reactions like RNA cleavage, splicing, and peptide bond formation. It provides examples of small self-cleaving ribozymes like the hammerhead, hairpin, VS, and HDV ribozymes. Larger RNA catalysts are discussed like RNase P and group I and II introns. Crystal structures of ribozymes like the hammerhead, hairpin, HDV, and group I introns are described. The peptidyl transferase activity of the ribosome is noted, with its crystal structure showing RNA is responsible for catalysis. In vitro selection has identified many nucleic acid catalysts for diverse

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 37

    RNA catalysis

    Outline
    • RNA transesterification
    • Naturally occurring catalysts
    • Catalytic functions
    • Catalytic mechanisms
    RNA transesterification
    • Exchange one phosphate ester for another
    • Free energy change is minimal (reversible)
    RNA transesterification
    • Nucleophile can be either the adjacent 2´ hydroxyl or
    another ester
    • Referred to as hydrolysis when water serves as the
    nucleophile
    RNA transesterification
    • Nucleophilic attack on the phosphorus center leads to a
    penta-coordinate intermediate
    • Ester opposite from the nucleophile serves as the leaving
    group (in-line attack)
    General mechanisms
    • Substrate positioning
    • Transition state stabilization
    • Acid-base catalysis
    • Metal ion catalysis
    RNA Catalysts
    Naturally occurring catalysts
    • RNA cleavage
    glmS ribozyme
    hammerhead ribozyme (crystal structure)
    hairpin ribozyme (crystal structure)
    Varkud satellite (VS) ribozyme (partial NMR structure)
    hepatitis delta virus (HDV) ribozyme (crystal structure)
    M1 RNA (RNase P) (partial crystal structure)

    • RNA splicing
    group I introns (crystal structure)
    group II introns
    *** U2-U6 snRNA (spliceosome) (partial NMR structure) ***

    • Peptide bond formation


    ribosome (crystal structure)
    Small self-cleaving ribozymes
    • Hammerhead, hairpin, VS, HDV ribozymes
    • Derivative of viral, viroid, or satellite RNAs
    • Involved in RNA processing during rolling circle
    replication
    • RNA transesterification via 2´ hydroxyl
    • Reversible: cleavage and ligation (excepting HDV)
    Hammerhead ribozyme
    • Three-stem junction with conserved loop regions
    • Coaxial stacking of stems II and III through
    extended stem II structure containing canonical
    Watson-Crick and non-canonical base pairs
    • Metal-ion catalysis
    Hammerhead ribozyme
    • In nature is self-
    cleaving (not a true
    enzyme)
    • Can be manipulated
    to function as a true
    catalyst
    • Biotechnological and
    potential therapeutic
    applications for
    target RNA cleavage
    Hammerhead ribozyme
    • Separation of catalytic and substrate strands
    • Strand with hairpin is the enzyme
    • Single strand is substrate
    • KM = 40nM; kcat = ~1 min-1;
    kcat/KM = ~107 M -1 min -1 (catalytic efficiency)
    • Compare to protein enzymes?
    RNA Catalysts
    • basics of catalytic reactions (cleavage)

    RNase A
    Protein
    enzyme

    Hammerhead
    ribozyme
    Hairpin ribozyme
    • In nature is part of a four-stem junction
    • Ribozyme consists of two stems with internal loops
    • Stems align side-by-side with 180 degree bend in
    the junction (hence ‘hairpin’)
    • Internal loops interact to form active site
    Hairpin ribozyme
    • Crystal structure
    reveals interactions
    between stems
    • Nucleobases position
    and activate sessile
    phosphodiester
    linkage
    • Combination of
    transition state
    stabilization and
    acid-base catalysis?
    HDV ribozyme
    • Genomic and
    antigenomic
    ribozymes
    • Nested
    pseudoknot
    structure
    • Very stable
    • Cleaves off 5´
    leader sequence
    HDV ribozyme
    HDV ribozyme
    • Active site positions an
    important cytidine near
    the sessile phophodiester
    bond
    • True enzyme RNase P
    • Cleaves tRNA precursor to
    generate the mature 5´ end
    • Composed of M1 RNA and C5
    protein (14 kD)
    • RNA is large and structurally
    complex
    • Protein improves turnover
    • Hydrolysis
    Group I introns
    • Large family of self-splicing introns usually
    residing in rRNA and tRNA
    • Two step reaction mechanism
    Group I intron structure
    • Crystal structure of ‘trapped’
    ribozyme before second
    transesterification reaction
    • Metal ion catalysis
    Group I intron structure
    Ribose zipper

    P1

    J8/7
    Group II introns
    Group II introns
    • Usually found in organelles
    (e.g. plant chloroplasts,
    mitochondria)
    • mechanism proceeds through a
    branched lariat intermediate
    structure which is produced by
    the attack of a 2’-OH of an
    internal A on the
    phosphodiester of the 5’-splice
    site

    • proteins thought to stabilize


    structure but not necessary for
    catalysis
    • no ATP or exogenous G needed
    Summary of splicing reactions
    The ribosome is a ribozyme
    • Ribosome is 2/3 RNA and 1/3 protein by mass
    • Crystal structures prove that RNA is responsible for
    decoding and for peptide bond formation
    Peptidyl transferase
    • Crystal structure of 50S subunit shows no protein within 20 Å
    of peptidyl transferase center
    • Closest component to
    aa-tRNA is adenosine
    2451 in 23S rRNA
    • Proposed acid-base
    mechanism for peptide
    bond formation
    • Recent evidence shows
    substrate positioning
    accounts for catalysis
    Prevalence of A-minor motifs

    Found 36 times in rRNA as type II/I couples


    Numerous isolated type I interactions
    RNA/DNA Catalysts
    RNA/DNA catalysis & evolution
    • in vitro selection
    RNA/DNA Catalysts
    RNA/DNA catalysis & evolution
    • increasing numbers of examples of reactions catalyzed by nucleic acids
    Table 1. Catalytic RNA and DNA molecules isolated from in vitro selection1

    Catalytic Nucleic Acid Reaction Catalyzed or Activity

    RNA Aminoacyl esterase


    RNA DNA Cleavage
    RNA RNA Cleavage
    RNA RNA Ligation
    RNA Isomerization of a bridged biphenyl
    RNA Self-phosphorylation
    RNA Amide bond cleavage
    RNA Aminoacylation
    RNA Alkylation
    RNA 5'-5' RNA ligation
    RNA Acyl transferase (ester and amide bond formation)
    RNA Porphyrin metalation with Cu2+ (heme biosynthesis)
    RNA Sulfur alkylation
    RNA 5'-self-capping
    RNA Carbon-carbon bond formation (Diels-Alder cycloaddition)
    RNA Amide bond formation
    RNA Peptide bond formation
    RNA Ester transferase

    DNA RNA cleavage


    DNA DNA ligation
    DNA Porphyrin metalation with Cu2+ (heme biosynthesis)
    DNA Cleave phosphoramidate bonds
    DNA DNA cleavage
    DNA Self-phosphorylation
    DNA 5'-self-capping
    1
    Ref. 44. This list is only an overview and does not include all nucleic acid catalysts
    discovered to date.
    DNA Catalysts
    DNA Catalysts
    Guanine Quartet Structures
    HDV ribozyme structure
    Proposed mechanism of catalysis
    pH (pD) profiles
    pH profiles (cation type)
    pH profiles (cation concentration)

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