Functional RNA - Introduction

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Functional RNA- Introduction

• Biochemistry 4000
• Dr. Ute Kothe

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Reading

• Biochemistry, Voet, 3rd edition
• Chapter 31-4. (Posttranscriptional Processing)
  32.2 (tRNAs)
• Reviews
• Wakeman et al., TIBS 2007 (riboswitches)
• Edwards et al., Curr Opin Struct Biol 2007
  (riboswitches)
• Scott, Curr Opin Struct Biol 2007 (ribozymes)
• Doudna Lorsch, Nat Struct Mol Biol 2005
  (ribozymes)

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Functional RNA - Classes

• Ribosomal RNA
• tRNA
• Spliceosomal RNA (small nuclear RNAs snuRNAs)
• Telomerase RNA
• RNA modification complexes small nucleolar RNA
  snoRNA
• Ribozymes
• Riboswitches
• microRNAs
• 4.5 S RNA (signal recognition particle)
• Etc.

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Primary Secondary Structure
Primary Structure Sequence of nucleotides in
(single-stranded RNA) Secondary
Structure Watson Crick base-pairing - can be
predicted by computer algorithms e.g. tRNA
cloverleaf structure
Yeast tRNAPhe
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RNA helices

• A-RNA
• resemble A-DNA
• wider an flatter right-handed helix than B-DNA
• 11.0 bp per turn
• pitch 30.9 Å
• base-pairs are inclined by 16.7º to the helix
  axis
• similar conformation is adopted by RNA-DNA hybrid

Voet, Chapter 29-1.
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Secondary structure elements
Hairpin (stem-loop)
Pseudoknot

• Bulge

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Tertiary Structure

• 3D structure
• Stabilized by tertiary interactions
• hydrogen bonds
• stacking interactions
• e.g. in tRNA tertiary base-pairs between D and T
  loop

Yeast tRNAPhe
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Tertiary Interactions in tRNA
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Non Watson-Crick Base-Pairs
If not constrained in a helix, basically every
edge of the nucleobase can participate in
base-pairing to another nucleobase.
Hoogsteen base-pairs compared to Watson-Crick
base-pairs
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RNA structural elements
A minor motif
U turn
Kissing Hairpins
GNRA tetraloop
K turn
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RNA Modifications

• about 100 different modifications known
• mainly base modification pseudouridine most
  common
• methylation of 2OH of ribose moiety
• individual pathway for each modification
• believed to stabilize RNA structure
• may modify base pairing (e.g. 5-oxyacetic acid
  in first anticodon position)

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RNA World Hypothesis

• Evolution of life may have started with RNA as
  the first biomolecule since RNA can store
  information (such as DNA) and catalyze reactions
  (such as proteins).

Evolution RNA Ribonucleoproteins Proteins
RNPs

• Limitations of RNA compared to proteins
• Few functional groups
• Low kcat
• Low stability

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Ribozymes

• Catalytic activity of RNA
• Peptide bond formation
• Phosphodiester cleavage
• RNA ligation
• Cyclic phosphate hydrolysis
• Limited polymerization of RNA
• RNA phosphorylation
• RNA aminoacylation
• Diels-Alder addition
• Glycosidic bond formation

Natural Ribozymes

• Artificial Ribozymes
• Generated by
• in vitro selection

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Ribozymes cleaving RNA

• Hairpin Ribozyme
• Hammerhead ribozyme
• Hepatitis Delta Virus Ribozyme (HDV)
• Varkud satellite ribozyme

• glms ribozyme
• RNase P
• (group I introns)
• (group II introns)

General Mechanism of Phosphodiester cleavage
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RNase A vs. HDV ribozyme
RNase A Acid-base catalysis by 2 His (for
details see Voet)
HDV ribozyme Acid-base catalysis by Cytidine
75 Involvement of a Mg2
What is the advantage of His over nucleobase for
acid-base catalysis?
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In vitro selected RNAs

• Aptamers small RNAs binding specific ligands
• Ribozymes small RNAs catalyzing desired
  reactions

• Usually less active than natural ribozymes
  (lower affinities, lower rate enhancements)
• due to limited number of evolution cycles

Diels Alder Ribozyme
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Riboswitch Regulators of Gene Expression

• Mainly found in prokaryotes, rarely in
  eukaryotes
• respond to various small molecules
• Control a large number of genes
• in 5 untranslated region (5 UTR)
• Evolutionary old simple control mechanism?

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Regulation types

• activation or repression
• transcriptional using a
• terminator hairpin
• or translational by
• sequestering the Shine-
• Dalgarno sequence

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Guanine and Adenine riboswitch

• Structurally Functionally very similar
• Highly selective
• Different regulation (activaiton vs. repression)
  of downstream genes

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Structure of Adenine Riboswitch

• Adenine binds at 3 helix junction
• Helices Pi P3 stack
• Loop 2 and Loop3 form tertiary interactions

• Binding pocket for Adenine
• specificity through Watson-Crick bp with U75
• hydrogen bonds also to sugar edge of adenine
• adenine deeply buried within the riboswitch

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RNA thermometer

• riboswitch regulating heat shock proteins
• at low temperatures, Shine-Dalgarno (grey box)
  sequences is sequestered by noncanonical
  base-pairing
• unfold at elevated temperatures release the
  Shine-Dalgarno sequence