Download The Versatility of RNA

The
Versatility
of RNA
Primary structure of RNA
•
RNA is a single strand molecule composed of
subunits called nucleotides joined by
phosphodiester bonds.
•
Each nucleotide subunit is composed of a
ribose sugar, a phosphate group, and a
nitrogenous base.
•
The common bases in RNA are adenine (A),
guanine (G), cytosine (C), and uracil (U).
PYRIMIDINES
PURINES
Six major types of RNA
RNA is involved in a wide range of cellular processes along the
pathways of gene expression, including DNA replication, RNA
processing, mRNA turnover, protein synthesis.
Some of the major types of
RNA:
Ribosomal RNA (rRNA) is an
essential component of the
ribosome.
Messenger RNA (mRNA) is a
copy of the genomic DNA
sequence that encodes a gene
product and binds to ribosomes in
the cytoplasm.
Transfer RNA (tRNA) is a small
RNA charged with a specific
amino acid. It delivers to the
ribosome the appropriate amino
acid.
Six major types of RNA
Small nuclear RNA (snRNA):
plays a role in pre-mRNA splicing,
a process which prepares the
mRNA for translation.
Small nucleolar RNA (snoRNA):
plays a role in RNA processing.
MicroRNA (miRNA): involved in
post-transcriptional gene
regulation;
each miRNA binds to
complementary sequence in a
target mRNA, usually resulting in
gene silencing, by triggering
degradation of mRNA or by
blocking translation by the
ribosome
Secondary structure
of RNA
Secondary structure of RNA
Secondary structure: single-stranded RNA molecules fold into a variety
of secondary structural motifs that are stabilized by both WatsonCrick and unconventional base pairing.
Helix formation occurs within one single-stranded chain of nucleotides.
The binding of divalent metal
ions (such as Mg2+, Ca2+, etc) to
the RNA is often critical to the
formation of a stable, folded
conformation because this ions
can shield the negative charge of
the RNA backbone, allowing
regions of the molecule to pack
more closely together.
Motifs include:
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Base-paired stems helix (with non canonical base pairs);
Hairpin (U-shaped) loops;
Internal loops
Bulges
Junction
Base-paired RNA adopts an
BaseA-type double helix
• The 2′-hydroxyl group hinders formation
of a B-type helix.
• A-type RNA helices (right-handed) with
Watson–Crick base pairs have a deep,
narrow major groove that is not well
suited for specific interactions with
ligands.
• The minor groove is shallow and broad
and includes the ribose 2′-OH groups
which are good hydrogen bond acceptors.
Because of these structural features, it is
common for RNA to be recognized by
RNA-binding proteins in the minor
groove.
Minor
Groove
Major
Groove
RNA helices often contain
nonnon
-canonical base pairs
• >20 different types of non-canonical (non-Watson-Crick) base pairs.
• Widen the major groove and make it more accessible to ligands or proteins.
• The most common of these are:
AGC and ACG base triples
•
RNA structure also frequently involve unconventional base pairing such as
base triples.
•
Typically involve one standard base pair.
•
The third base interacts in a variety of unconventional ways.
Do these unusual base interactions have any
functional significance?
Yes ! Non canonical base pairs and base triples are
important mediators of:
• RNA self-assembly.
• RNA-protein interactions.
• RNA-ligand interactions.
(e.g., widen the major groove making
it more accesible to ligande)
tRNA structure
•
tRNA is transcribed as a molecule about twice as long as its final form. The
pre-tRNA transcript is then cut at both the 5′ and 3′ ends.
•
The average tRNA is about 76 nt long.
•
All tRNAs fold into the same general shape.
Cloverleaf secondary structure
L-shaped tertiary structure
tRNA structure
•
More than 50 modified bases in tRNA. Modifications
include simple methylation to complete restructuring of
the purine ring.
For example:
Methylation:
substitution of an
atom or group by a
methyl group (CH3)
tRNA structure
Inosine (I) was the
first modified
nucleoside to be
identified in tRNA
Pseudouridine (ψ) was
the first identified in
any RNA.
tRNA loops each have a separate function
tRNA has the sequence ACC
on the 3’ to which the amino
acid is attached
D loop
recognition by the
aminoacyl tRNA
synthetases that
charge the
appropriate amino acid
T-loop
Involved in
recognition by
the ribosome.
Anticodon loop
base pairs with the mRNA
codon
Tertiary structure
of RNA
Common tertiary structure motifs in RNA
PSEUDOKNOTS MOTIF
A pseudoknot motif forms when a single-stranded loop base pairs
with a complementary sequence outside this loop and folds into a
three-dimensional structure by coaxial stacking. The RNA appears to be
tied in a knot.
For example, the RNA component of Telomerase has a highly conserved
pseudoknot that is essential for its function.
Common tertiary structure motifs in RNA
TETRALOOP MOTIF
A stem loop with the tetraloop
sequence “UUUU” is particulary
stable due to special base-stacking
interaction in the loop
RIBOSE ZIPPER MOTIF
This motif stabilizes helix-helix interactions
between the minor groove. The interactions
involve hydrogen bonding between the 2'-OH
of a ribose in one helix and the 2-oxygen of a
pyrimidine base of the other helix.
Common tertiary structure motifs in RNA
KISSING HAIRPIN LOOP
Two hairpin loop forms a kissing interaction by base pairing
between single-stranded regions of two hairpin loops with
complementary sequences.
Two Coaxial stacked arms form the familiar
L-shape of tRNA
Coaxial stacking, or helical stacking,
occurs when the nucleotide bases
from two separate base-paired
stems stack and align to form what
appears as a continuous helix.
7 bp acceptor stem in tRNA stacks
on the 5 bp T stem to form an Atype helical arm.
The D stem and anticodon stem
stack to form a second helical arm
How do these complicated structure get folded properly in the cell???
Ribozymes
Classic definition of an enzyme:
An enzyme increases the rate, or velocity, of a chemical reaction
without itself being changed in the overall process. Enzymes reduce
the activation energy, Ea, of a reaction.
Activation energy, Ea: minimum energy that must be input to a
chemical system in order for a chemical reaction to occur.
Ribozymes
RIBOZYMES
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RNA molecules with catalytic activity. Naturally occurring ribozymes
can be autocatalytic, which lead to their own modification, or they can
be true enzymes.
Form substrate-binding sites.
Lower the activation energy.
Allow reaction to proceed much faster.
Mode of ribozyme action
Some ribozymes use a two-metal-ion mechanism for catalysis.
(Binding of divalent cations (Mg2+ ) in the active site is critical for
their
folding into an active state)
•
• Other ribozymes use general acid-base chemistry, by donating or
accepting protons during chemical step of reaction.
Naturally occurring ribozymes
Two different groups based on difference in size and reaction
mechanism:
Small ribozymes
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Vary in size from 40 to 154 nucleotides.
Cleave RNA to generate a 2′-3′-cyclic phosphate and a product with a
5′-OH terminus.
This group of small ribozymes includes
The hammerhead, so called for its three helices in a T shape, is the most frequently
found catalytic motif in plant phatogenic RNAs called virions.
Naturally occurring ribozymes
Large ribozymes
• Vary in size from a few hundred to 3000 nucleotides.
• Cleave RNA to generate 3′-OH termini.
• RNA components of:
Evidence for the RNA world hypothesis
•
Life first existed in the form of
replicating RNA molecules.
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RNA has all the structural
prerequisites for self-replication.
•
RNA genomes are widespread
among viruses.
•
RNA molecules:
Self-fold into 3-D structures.
Recognize other macromolecules
and ligands with precision.
Catalyze a diversity of reactions.
•
The ribosome is a ribozyme.
Compared with DNA or protein, RNA is clearly the most selfsufficient molecule. RNA molecules are capable of doing basically all
that proteins can do.
Evidence for the RNA world hypothesis
Later, during a
hypothetical transitional
period, RNA catalyzed
the synthesis of
proteins and these
proteins catalyzed the
transition from RNA to
DNA.
The Discovery of Reverse
Transcriptase
Howard Temin
• Studied RNA tumor viruses
most of his life
• His early days he spent time
working on Rous sarcoma
virus (RSV)
Howard Temin Hypothesis
“The RNA genome of RSV is
converted into a DNA provirus.”
Howard Temin Hypothesis
“RSV is sensitive to inhibitors of
DNA synthesis and suggesting that
DNA, complementary to the RSV
genomic RNA, is present in
transformed cells.”
David Baltimore
• Spent time studying virus
replication
• He looked at RNA and DNA
at a more biochemical view
looking deep within
individual virions
• He later focused on RNA
tumor viruses and concluded
with Rauscher murine
leukemia virus (R-MLV)
Experiment
•They disrupted their pure stock viruses by using
nonionic detergents
•“Can a retrovirus perform DNA synthesis?”
•Then “radiolabeled deoxthymidine triphosphate
(dTTP) along with three deoxynucleotide
triphosphates (dATP, dCTP, dGTP) to the virion
preparation.”
Did these reactions contain any incorporation of
radioactive dTTP into DNA?
YES!
Each experiment, dTTP turned into nucleic acid
RNA secondary structure
Pseudoknot
Hairpin loop
Duble helix
Internal loop
Bulge loop
Multibranch loop
(helical junction
junction))
Probes
Target
Detection
DMS
A(N1)>>C(N3)
CMCT
U(N3)>>G(N1)
Primer extension
T1 RNase
Unpaired G
5’-end and 3’-end
labelling
T2 RNase
Unpaired
A>C,U,G
5’-end and 3’-end
labelling
V1 RNase
Paired or stacked N
5’-end and 3’-end
labelling
Primer extension
ladder
∆G
10°
10°C
C T1 T2 V1
20°
20°C
C T1 T2 V1
37°
37°C
C T1 T2 V1
strong T1 and T2 cuts
weak T1 and T2 cuts
strong V1 cuts
weak V1 cuts