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RNA structure and function summary
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- Instelling
Summary of the course RNA structure and function, with information highlighted that was important for the exam and the resit.
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Voorbeeld 3 van de 23 pagina's
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Week 1Basics
Main differences between RNA and DNA are that RNA is synthesized as single stranded
and DNA as double stranded. RNA has a ribose sugar and DNA a deoxyribose sugar. The
ribose is an OH group and the deoxyribose is an H group. Main difference between DNA and
RNA is the presence or absence of the hydroxyl group at the 2’ position. RNA has a uracil
(U) and DNA has a thymidine (T). RNA is made of sugar, nucleotide, phosphates at 5’ end.
Adenine and guanine are purines
Uracil, thymine and cytosine are pyrimidines
The ribose is different between RNA and DNA, while the bases are not
different, for the exception of U or T being present.
In RNA there are also non-standard nucleotides: dihydrouridine, inosine,
ribothymidine, pseudouridine. Also DNA can be methylated for epigenetics,
while RNA can’t. Inosine is mostly found in the anticodon region in for
example tRNA.
Primary structure RNA molecule is a right handed helical conformation
dominated by base-stacking interactions.
RNA is unstable under alkaline conditions. Hydrolysis is also catalyzed by
enzymes like ribonuclease and RNase. When RNA is made and it fulfills his function, RNA is
degraded. RNase enzymes around us that are abundant:
- S-RNase in plants that prevent inbreeding
- RNase P is a ribozyme (enzyme made of RNA) that processes tRNA precursors
- Dicer is an enzyme that cleaves double-stranded RNA into oligonucleotides to
protect from viral genomes and RNA interference
- RNA exosome is a ubiquitous complex of 3’-5’ exoribonucleases
Double stranded helices in nucleic acids: DNA adopts a B-form helices
and RNA mostly adopts the A-form helices. The B-form helix has
wide/open major grooves and small minor grooves. The A-form helix has
deep and narrow major grooves and the minor groove is wider. The
bases indicated in purple are more on the surface of the helix. Also the
base pairs are more compact in the A-form helix.
So for interactions with DNA the major groove is more important and for
interactions with RNA the minor groove is more important. The A-form
and B-form helix are right handed and the Z-form helices are left handed.
,Secondary structure of RNA
Internal loop asymmetric: unequal
number of non-base paired
residues in both strands and
symmetric: equal number of
non-base paired residues in both
strands
Bulge single nucleotide bulge and
multiple nucleotide bulge
There are more RNA secondary structures in the untranslated
regions of mRNA, so in the 5’ UTR and 3’ UTR end. This is
determined by an experiment called SHAPE.
Unusual base pairing of RNA is G-U, called wobble-base pairing.
Watson-Crick base-pairing is G-C and A-U. Other examples of
unusual base-pairing are A-A, G-G, U-U, C-C, A-G and C-U.
Hoogstein base-pairing is of a purine and a pyrimidine, like TAT or
CGC.
Tertiary structures of RNA
Strategies for assembling RNA tertiary structures
- Long-range base-pairing
- Coaxial stacking is short helices are often
stacked on top of each other
- Hydrogen bonding are base triples (Hoogstein base-pairing), ribose zippers
(involvement of ribose 2’-OH groups in hydrogen bonding)
- Metal ions are positively charged metal ions help negatively charged RNA strands to
associate into helices and also to bind to specific sites within RNA tertiary
structures to hold RNA helices together
A minor interaction: adenines forming tertiary contacts with the minor grooves of
distal helices. →
RNA quadruplex structures: Repeated stretches of guanine-rich sequences
can form quadruple base pairs that stack on each other to form quadruplexes.
Ribose zippers: The 2′-hydroxyl group of the RNA backbone is a stabilizing
component in many tertiary interactions, since it can make two hydrogen bonds,
acting as both hydrogen-bond donor and acceptor.
RNA tertiary structures are stabilized by the binding of metal ions
It is generally believed that physiological concentrations of monovalent ions can
promote secondary structure formation but that magnesium ions are required for
stabilization of RNA tertiary structure. However, there are many different roles for
monovalent and divalent ions in RNA structure. Magnesium can be associated quite loosely
with RNA molecules, because magnesium is still hydrated.
, Methodologie
Abundance of RNA is mRNA<tRNA<rRNA and the most convenient way of isolating mRNA
out of total RNA by using the 3’ poly A-tail of mRNA. Three technologies to assay the
expression level of an individual gene are RT-qPCR, Northern blot and primer extension.
RNA-seq can be used to quantify gene expression, alternative splicing and determine fusion
genes.
RNA isolation and quality check
Most mRNAs have a poly(A) tail, while structural RNAs don’t. RNA isolation from total RNA
can be done by oligo(dT) selection, where non-polyadenylated RNA gets separated from
polyadenylated RNA. Total RNA is denatured to expose the poly(A) tails. Poly(A)-containing
RNA is then bound to oligo(dT) cellulose, with the remainder of the RNA washing through.
The poly(A) + RNA is eluted by removing salt from the solution, thus destabilizing the dT:rA
hybrid.
RNA is denatured at relatively high temperatures and in the presence of urea and chemicals
that trap divalent metal ions (EDTA).
Agarose/polyacrylamide gel electrophoresis
Separation of nucleic acids can be based upon size and charge. The phosphate groups of
the nucleotides that make up (fragments of) DNA and RNA give them a negative charge.
Thus, when an electric current is applied, the DNA/RNA (fragments) move(s) to the positive
pole. Intercalating dyes (ethidium bromide; SYBR Green) can be used to visualize
the separated nucleic acids in the gel. Most frequently applied gel systems:
- Agarose gels: polysaccharide derived from red seaweed and DNA test gel for RNA
integrity (0.8-1.2% agarose)
- Polyacrylamide gels: acrylamide chemically crosslinked to bisacrylamide and small
RNA molecules 50-500 nucleotides
Gene quantification of individual genes
Reverse transcription-PCR (quantification)
Depending on the (selection of) RNA alternative oligonucleotide primers can
be used for reverse transcription. A mixture of random hexamers will initiate
cDNA synthesis at any RNA. The product is amplified in a PCR reaction using
a thermostable DNA polymerase, such as Taq polymerase.
If you want specification in the first step, you can use specific primers.
Northern blot hybridization (quantification + size)
Northern blot is used to visualize amounts of RNA in samples. First RNA will
be isolated and then separated by gel electrophoresis. It is transferred to some
electrostatically interacting surface, such as nitrocellulose or nylon. The RNA backbone
binds (electrostatically) to the surface; note that the bases are still exposed for hydrogen
bonding when the RNA is bound. The sheet of nitrocellulose or nylon is then either heated or
exposed to ultraviolet light in order to permanently attach the RNA to its surface.
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