Download Functional RNA

Functional RNA
- Introduction
Biochemistry 4000
Dr. Ute Kothe
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)
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.
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
RNA helices
A-RNA
• resemble A-DNA
• wider an flatter righthanded 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.
Secondary structure elements
Hairpin
(stem-loop)
Bulge
Pseudoknot
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
Tertiary Interactions in tRNA
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
RNA structural elements
A minor motif
U turn
Kissing Hairpins
K turn
GNRA tetraloop
RNA
Modifications
• about 100 different
modifications known
• mainly base modification:
pseudouridine most
common
• methylation of 2’OH 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)
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
RNPs
Proteins
Limitations of RNA
compared to proteins:
• Few functional groups
• Low kcat
• Low stability
Ribozymes
Catalytic activity of RNA:
• Peptide bond formation
• Phosphodiester cleavage
Natural Ribozymes
• RNA ligation
• Cyclic phosphate hydrolysis
• Limited polymerization of RNA
• RNA phosphorylation
• RNA aminoacylation
• Diels-Alder addition
• Glycosidic bond formation
Artificial Ribozymes
-Generated by
in vitro selection
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:
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?
In vitro selected RNAs
1. Aptamers – small RNAs binding specific ligands
2. 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
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?
Regulation types
• activation or repression
• transcriptional using a
terminator hairpin
• or translational by
sequestering the ShineDalgarno sequence
Guanine and Adenine riboswitch
•Structurally &
Functionally very
similar
•Highly selective
•Different regulation:
(activaiton vs.
repression) of
downstream genes
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
RNA thermometer
• riboswitch regulating heat
shock proteins
• at low temperatures, ShineDalgarno (grey box)
sequences is sequestered
by noncanonical basepairing
• unfold at elevated
temperatures & release the
Shine-Dalgarno sequence