RNA Structure and Function
(KW1 V)
September 2021 – November 2021
1. Lecture 1 – Introduction
2. Lecture 2 – RNA Methodologies
3. Lecture 3 – RNA structure prediction
4. Lecture 4 – small non-coding RNA
5. Lecture 5 – siRNA pathway (RNA interference)
6. Lecture 6 – RNA viruses
Lecture 6.2 – CRISPR Cas
7. Lecture 7 – Long noncoding RNA
Lecture 7.2 – Long noncoding RNA: Functions and typical examples
8. Lecture 8 – RNA therapeutics - Therapeutic RNAs
,RNA structure and function – summary
Lecture 1
The molecule's ribonucleotide building blocks are made up of three parts: a sugar molecule, a
phosphate group at 5´ and one of the four bases that form the alphabet of RNA's genetic code —
adenine, uracil, cytosine and guanine.
DNA contains the sugar deoxyribose, while RNA contains the sugar ribose. DNA uses the bases adenine,
thymine, cytosine, and guanine; RNA uses adenine, uracil, cytosine, and guanine. Uracil differs from
thymine in that it lacks a methyl group on its ring.
Building blocks of RNA: non-standard nucleotides
In RNA, other nucleotides are frequently found. In dihydrouridine, one double bond is replaced by a
regular bond, and two hydrogens are added. Pseudouridine is chemically the same as uridine, however
the base is attached to the ribose differently. It is linked via a carbon, while usually a nitrogen is present
instead. Inosine can base pair with three nucleotides. It is not necessary to have 64 different tRNAs to
recognize all codons.
“The wobble position of a codon refers to the 3rd nucleotide in a codon. Binding of a codon in an mRNA
the cognate tRNA is much "looser" in the third position of the codon. This permits several types of non-
Watson–Crick base pairing to occur at the third codon position. The four main wobble base pairs are
guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-
cytosine (I-C).”
Non-standard nucleotides are
mostly found in non-coding
mRNAs like tRNA p.e. mRNA is
supposed to be free of non-
standard nucleotides as it is
coding mRNA, however, it was
discovered that they can still
contain them.
,Primary structure of RNA
From a DNA template, the RNA strand
is added from 5´to 3´ to the growing
strand. Phosphodiester bonds are
generated. The single RNA strand
tends to assume a right-handed helical conformation
dominated by base-stacking interactions. The bases
visible in yellow are present on top of each other, as the
pi-stacking is energetically favourable, which leads to
the helical structure.
(Recognizing orientation of a helix: Take your right hand
and slide it down the helix, if the thumb goes to the right
side, it is a right-handed helix)
Hydrolysis of RNA
RNA is unstable under alkaline conditions, the molecules can quickly be degraded when the pH is
raised, the phosphodiester bonds are disrupted. The pH is not relevant for biological systems, so the
hydrolysis of RNA is also catalysed by enzymes like ribonucleases, and RNases. There is a continuous
synthesis and degradation of RNA. RNase enzymes are abundant around us, S-RNase in plants prevents
inbreeding, RNase P is a ribozyme (enzyme made of RNA) that processes tRNA precursors. A Dicer is
an enzyme that cleaves double-stranded RNA into oligonucleotides, which protects the cell from viral
genomes, and is used for RNA interference. RNA exosome is a ubiquitous complex of 3’-5’
exoribonucleases, which starts at the 3´end of a single strand, and cleaves off nucleotides one by one
into the 5´direction.
Base-catalyzed hydrolysis of RNA
RNA is sensitive to alkaline conditions. The
hydroxyl base can take off the hydrogen at the
2´position of the RNA molecule. The electrons
attack the phosphate, which leads to the
formation of a cyclic phosphate,
simultaneously the phosphodiester bond will
be disrupted. This results in two products, with
a cleavage at the phosphodiester between the
nucleotides.
Secondary and tertiary structure of RNA
In a single RNA strand are multiple regions of bases that are
complementary to same molecule. Base pairing occurs fast after
synthesis, and is energetically favourable. This leads to secondary
structures like hairpin or stem-loop (larger than hairpin, depends on
number of nucleotides) formation, or tertiary structures like the
pseudoknot.
, By complementary base pairing, double stranded
regions are formed. The RNA helix is not
structurally identical to the DNA helix, which is
most commonly present in the b-form, while RNA
is present in the a-form. A hydroxyl residue is
present on every ribose on the backbone of the
RNA, which leads to different interactions and
makes the a-form the most stable one. The a-
form is angled at 20°, while the b-form is angled
at 6° between the base pairs. (In in the a-form) In
the minor groove, the base pairs are closer to the
surface and easily accessible (for proteins for
interactions e.g.), the major groove on the other
hand is much deeper. This suggests, that
interactions with double stranded RNA must depend on the bases, such interactions are most likely in
the minor groove. The diameter of the a-form is somewhat larger than the b-form.
Secondary structure of RNA
In the internal loop, the asymmetric formation shows an unequal number of none-base paired residues
in both strands, while the symmetric internal loop shows an equal number of non-base paired residues
in both strands. Single nucleotide bulges and multiple nucleotide bulges are possible. All these
structures can be seen combined in large molecules.
The majority of base pairs interact, and are paired in e.g. rRNA, however, this is not the case in mRNA,
the presence of a secondary and tertiary structure would make it difficult for the ribosome to read the
mRNA. A technique called SHAPE cane be used to detect the presence of structures, which showed
that both 3´and 5´UTR have more structures compared to the coding sequences.
Phylogenetic conservation of base-pairing
Support for secondary structures can also be obtained from phylogenetic data. The RNA component
of RNase P is involved in the process of the tRNA and therefore found in all organisms, however, there
are slight differences in the sequence and structure. When they are compared for a whole series of
organism, it gives information on the residues that are most conserved. Evolutionary conservation
shows that it is important for either structure or function. This gives support for the presence of
secondary structures. If a nucleotide replacement in a single stranded RNA does not hurt the function
(KW1 V)
September 2021 – November 2021
1. Lecture 1 – Introduction
2. Lecture 2 – RNA Methodologies
3. Lecture 3 – RNA structure prediction
4. Lecture 4 – small non-coding RNA
5. Lecture 5 – siRNA pathway (RNA interference)
6. Lecture 6 – RNA viruses
Lecture 6.2 – CRISPR Cas
7. Lecture 7 – Long noncoding RNA
Lecture 7.2 – Long noncoding RNA: Functions and typical examples
8. Lecture 8 – RNA therapeutics - Therapeutic RNAs
,RNA structure and function – summary
Lecture 1
The molecule's ribonucleotide building blocks are made up of three parts: a sugar molecule, a
phosphate group at 5´ and one of the four bases that form the alphabet of RNA's genetic code —
adenine, uracil, cytosine and guanine.
DNA contains the sugar deoxyribose, while RNA contains the sugar ribose. DNA uses the bases adenine,
thymine, cytosine, and guanine; RNA uses adenine, uracil, cytosine, and guanine. Uracil differs from
thymine in that it lacks a methyl group on its ring.
Building blocks of RNA: non-standard nucleotides
In RNA, other nucleotides are frequently found. In dihydrouridine, one double bond is replaced by a
regular bond, and two hydrogens are added. Pseudouridine is chemically the same as uridine, however
the base is attached to the ribose differently. It is linked via a carbon, while usually a nitrogen is present
instead. Inosine can base pair with three nucleotides. It is not necessary to have 64 different tRNAs to
recognize all codons.
“The wobble position of a codon refers to the 3rd nucleotide in a codon. Binding of a codon in an mRNA
the cognate tRNA is much "looser" in the third position of the codon. This permits several types of non-
Watson–Crick base pairing to occur at the third codon position. The four main wobble base pairs are
guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-
cytosine (I-C).”
Non-standard nucleotides are
mostly found in non-coding
mRNAs like tRNA p.e. mRNA is
supposed to be free of non-
standard nucleotides as it is
coding mRNA, however, it was
discovered that they can still
contain them.
,Primary structure of RNA
From a DNA template, the RNA strand
is added from 5´to 3´ to the growing
strand. Phosphodiester bonds are
generated. The single RNA strand
tends to assume a right-handed helical conformation
dominated by base-stacking interactions. The bases
visible in yellow are present on top of each other, as the
pi-stacking is energetically favourable, which leads to
the helical structure.
(Recognizing orientation of a helix: Take your right hand
and slide it down the helix, if the thumb goes to the right
side, it is a right-handed helix)
Hydrolysis of RNA
RNA is unstable under alkaline conditions, the molecules can quickly be degraded when the pH is
raised, the phosphodiester bonds are disrupted. The pH is not relevant for biological systems, so the
hydrolysis of RNA is also catalysed by enzymes like ribonucleases, and RNases. There is a continuous
synthesis and degradation of RNA. RNase enzymes are abundant around us, S-RNase in plants prevents
inbreeding, RNase P is a ribozyme (enzyme made of RNA) that processes tRNA precursors. A Dicer is
an enzyme that cleaves double-stranded RNA into oligonucleotides, which protects the cell from viral
genomes, and is used for RNA interference. RNA exosome is a ubiquitous complex of 3’-5’
exoribonucleases, which starts at the 3´end of a single strand, and cleaves off nucleotides one by one
into the 5´direction.
Base-catalyzed hydrolysis of RNA
RNA is sensitive to alkaline conditions. The
hydroxyl base can take off the hydrogen at the
2´position of the RNA molecule. The electrons
attack the phosphate, which leads to the
formation of a cyclic phosphate,
simultaneously the phosphodiester bond will
be disrupted. This results in two products, with
a cleavage at the phosphodiester between the
nucleotides.
Secondary and tertiary structure of RNA
In a single RNA strand are multiple regions of bases that are
complementary to same molecule. Base pairing occurs fast after
synthesis, and is energetically favourable. This leads to secondary
structures like hairpin or stem-loop (larger than hairpin, depends on
number of nucleotides) formation, or tertiary structures like the
pseudoknot.
, By complementary base pairing, double stranded
regions are formed. The RNA helix is not
structurally identical to the DNA helix, which is
most commonly present in the b-form, while RNA
is present in the a-form. A hydroxyl residue is
present on every ribose on the backbone of the
RNA, which leads to different interactions and
makes the a-form the most stable one. The a-
form is angled at 20°, while the b-form is angled
at 6° between the base pairs. (In in the a-form) In
the minor groove, the base pairs are closer to the
surface and easily accessible (for proteins for
interactions e.g.), the major groove on the other
hand is much deeper. This suggests, that
interactions with double stranded RNA must depend on the bases, such interactions are most likely in
the minor groove. The diameter of the a-form is somewhat larger than the b-form.
Secondary structure of RNA
In the internal loop, the asymmetric formation shows an unequal number of none-base paired residues
in both strands, while the symmetric internal loop shows an equal number of non-base paired residues
in both strands. Single nucleotide bulges and multiple nucleotide bulges are possible. All these
structures can be seen combined in large molecules.
The majority of base pairs interact, and are paired in e.g. rRNA, however, this is not the case in mRNA,
the presence of a secondary and tertiary structure would make it difficult for the ribosome to read the
mRNA. A technique called SHAPE cane be used to detect the presence of structures, which showed
that both 3´and 5´UTR have more structures compared to the coding sequences.
Phylogenetic conservation of base-pairing
Support for secondary structures can also be obtained from phylogenetic data. The RNA component
of RNase P is involved in the process of the tRNA and therefore found in all organisms, however, there
are slight differences in the sequence and structure. When they are compared for a whole series of
organism, it gives information on the residues that are most conserved. Evolutionary conservation
shows that it is important for either structure or function. This gives support for the presence of
secondary structures. If a nucleotide replacement in a single stranded RNA does not hurt the function
