RNA Characteristics, stability and forms 1

MLAB 230 - Molecular Biology

Topic 4:
RNA characteristics, stability and forms
 RNA Characteristics, stability and forms 2

RNA vs DNA
➢ At first glance, RNA appears very similar to DNA. RNA chemical composition
only differs from that of DNA in supposedly minor aspects.

➢ RNA differs from DNA in three main features:

1. The backbone of RNA contains ribose rather than 2’-deoxyribose

Ribose has a hydroxyl group at the 2’ position in

place of a hydrogen atom
 RNA Characteristics, stability and forms 3

RNA vs DNA
➢ At first glance, RNA appears very similar to DNA. RNA chemical composition
only differs from that of DNA in supposedly minor aspects.

➢ RNA differs from DNA in three main features:

2. RNA contains uracil in place of thymine

Thymine = 5-methyl-uracil

• Uracil has the same single-ringed structure

as thymine, except that it lacks the methyl
group at position 5

• Like Thymine, Uracil pairs with Adenine

 RNA Characteristics, stability and forms 4

RNA vs DNA
➢ At first glance, RNA appears very similar to DNA. RNA chemical composition
only differs from that of DNA in supposedly minor aspects.

➢ RNA differs from DNA in three main features:

3. RNA is usually found as a single polynucleotide chain

mRNA has the same sequence as the non-template strand
(also known as the coding strand)

• RNA is copied as a single strand off only one

of the two strands of the DNA template, and
its complementary strand does not exist.

• RNA is capable of forming long double

helices, but these are unusual in nature.

two strands are more stable than one

DNA>RNA
 RNA Characteristics, stability and forms 5

The history of RNA

➢ The discovery of RNA began with the discovery of nucleic acids by Friedrich
Miescher in 1868 who called the material 'nuclein' since it was found in the
nucleus.

➢ Between 1890 and 1930, RNA was found to be chemically different from
DNA.

➢ The role of RNA in protein synthesis was discovered in 1939.

➢ For many years, RNA was seen as simply playing a supporting role to DNA in
information transfer.

➢ We now know that RNA is far richer and more intricate in structure than DNA
and far more versatile in function than first appreciated.
 RNA Characteristics, stability and forms 6

RNA as the viral genetic material

➢ All organisms and many viruses have DNA as their genetic material.
However, some bacteriophages, a number of animal viruses and a number of
plant viruses have RNA as their genetic material.

➢ Depending on the virus, the genetic material may be double stranded RNA,
or single-stranded RNA.

➢ No known prokaryotic or eukaryotic organism has RNA as its genetic

material.

➢ More RNA functions to come…

 RNA Characteristics, stability and forms 7

RNA structural characteristics

• The secondary structures of RNA

➢ Although RNA is single-stranded, these molecules often exhibit many double-

helical characters.
➢ RNA chains frequently fold back on themselves to form base-paired
segments between short stretches of complementary sequences.

➢ If the two stretches of complementary sequence are near each other, the
RNA may adopt a stem-loop structure.

➢ Double-helical RNA may also exhibit internal loops when unpaired

nucleotides are found on either side of the stem.

➢ Bulges are formed when an unpaired nucleotide is found on one strand of

the RNA double helix.

➢ Junctions are important structural elements that form when three or more
helices come together.
 RNA Characteristics, stability and forms 8

RNA structural characteristics

• The secondary structures of RNA

Stem
 RNA Characteristics, stability and forms 9

RNA structural characteristics

• The Pseudoknot
➢ Base pairing can also take place between sequences that are not
contiguous to form complex structures named pseudoknots.
 RNA Characteristics, stability and forms 10

RNA structural characteristics

• Non conventional base pairings

➢ Interaction between two bases of a nucleic acid other than the

standard base pairings

➢ There are three main types of non-canonical base pairs:

• those stabilized by polar hydrogen bonds
• those having interactions among C− H and O/N groups
• those that have hydrogen bonds between the bases themselves

➢ For example the G:U base pair, which has hydrogen bonds between
N3 of uracil and the carbonyl on C6 of guanine, and between the
carbonyl on C2 of uracil and N1 of guanine dnt memorize
 RNA Characteristics, stability and forms 11

RNA structural characteristics

• Non conventional base pairings

➢ These base pairings can be found in all combinations in RNA (GA and GU
are the most abundant in ribosomal RNA)

➢ Thus RNA chains have an enhanced capacity for self-

complementarity
 RNA Characteristics, stability and forms 12

RNA structural characteristics

• 2’ Hydroxyl group in the backbone of RNA

➢ Prevents RNA from adopting a B-form helix. Rather, double-helical

RNA resembles the A-form structure of DNA.

➢ Can chemically attack the adjacent phosphodiester bond to cleave

the backbone in regions not involved in double helix formation.

• Tertiary structures
➢ Further unconventional base pairing favors complex folding of RNA
➢ Can be assisted by proteins (ribosome).

These features of RNA increase its stability and protect

it against RNAses and hydrolysis
 RNA Characteristics, stability and forms 13

RNA Forms and functions

➢ In both prokaryotes and eukaryotes, there are three main types of
RNA involved in protein synthesis :

• Messenger RNA (mRNA) role of the template

• Ribosomal RNA (rRNA) role of machine
• Transfer RNA (tRNA) role of bringing aa to growing chain

➢ Other types of RNAs are :

• snRNA (splicing of pre-mRNA) small nuclear RNA

• snoRNA (nucleotide modification) small nucleolar RNA --editing: example modify
nucleotides to form new ones like
• miRNA, siRNA, shRNA (gene regulation) I, psi... (slide 22)
microRNA
• lncRNA (epigenetic regulation) small interference RNA
long non-coding RNA short hairpin RNA

genetic regulation: sequence; mutations/ repressors...

epigenetic regulation: same sequence --> diff phenotype (Ex- methylation: shutting down/silencing) / not repressor
 RNA Characteristics, stability and forms 14

Messenger RNA (mRNA)

➢ The only protein-coding form represents 5% of all RNAs
➢ The mature, biologically active mRNA in both prokaryotic and
eukaryotic cells has three main parts :

• A 5’ Untranslated region (5’UTR) or leader sequence

• A protein-coding sequence, which specifies the amino acid sequence
of a protein during translation
• A 3’ Untranslated region (3’UTR) or trailer sequence

AUG stop
codons
 RNA Characteristics, stability and forms 15

Messenger RNA (mRNA)

➢ mRNA production is different in bacteria and eukaryotes

• In bacteria the RNA transcript functions directly as the mRNA

molecule and the lack of nucleus allows the bacteria to directly
translate the mRNA while it is being transcribed = coupled
transcription-translation
in eukaryotes, mRNA needs to be protected (in prokaryotes --> less exposure)

• In eukaryotes, the RNA transcript (the pre-mRNA) is modified in the

nucleus by RNA processing mechanisms to produce the mature
mRNA.
• The mRNA needs to migrate from the nucleus to the cytoplasm
thus it needs to be completely transcribed before being translated.

• Bacterial mRNAs are polycistronic (coding for multiple genes) while

eukaryotic mRNAs are monocistronic.
 RNA Characteristics, stability and forms 16

Messenger RNA (mRNA)

many types polyribosome:

of proteins many copies
1 type of protein
 RNA Characteristics, stability and forms 17

Messenger RNA (mRNA)

➢ The age of Intron
• Before 1977 it was believed that the mRNA is a true copy of his
DNA

• In 1977 two molecular biologists, Richard Roberts and Phil Sharp,

discovered that genes of eukaryotic cells (such as those of
animals, plants and fungi) exhibit many places where the coding
information is interrupted.

• The mRNA molecules from eukaryotic cells were significantly

shorter than their corresponding gene sections. The mRNA, which
reached the cytoplasm, exhibited large gaps, compared to the
corresponding DNA.
 RNA Characteristics, stability and forms 18

Messenger RNA (mRNA)

➢ The age of Intron
• Electron microscopy of DNA/RNA hybrid of adenovirus mRNA

interruptions

sections of DNA not found in RNA

 RNA Characteristics, stability and forms 19

Messenger RNA (mRNA)

➢ The age of Intron
• In eukaryotes in general, protein-coding genes typically have non-
amino acid–coding sequences called introns (intragenic regions)
between the other sequences that are present in mRNA, the exons

• Introns are important for gene expression levels and enhancement/

repression of genes.
 RNA Characteristics, stability and forms 20

Transfer RNA (tRNA)

➢ During translation of mRNA, each transfer RNA (tRNA) brings a
specific amino acid to the ribosome to be added to a growing
polypeptide chain.

➢ The correct amino acid sequence of a polypeptide is achieved as a

result of:
• (1) the binding of each amino acid to a specific
tRNA
• (2) the binding between the codon of the mRNA and the
complementary anticodon in the tRNA.

➢ Eukaryotic tRNA genes are repeated many times in the genome and
produce precursor tRNA (pre-tRNA) molecules, each of which has
extra sequences at each end that are removed post-transcriptionally.
 RNA Characteristics, stability and forms 21

Structure of tRNA
➢ 75 to 90 nucleotides long, each type having a different sequence that
defines the specific amino acid binding.

➢ Complementary base pairing in tRNA induce a Cloverleaf-like

structure.

➢ 4 stems separated by 4 loops I, II, III and IV.

➢ Loop II contains the three-nucleotide anticodon sequence.

➢ Codon–anticodon pairing is crucial to the addition of the amino acid

specified by the mRNA to the growing polypeptide chain.
 RNA Characteristics, stability and forms 22

Structure of tRNA
➢ All tRNA molecules have the
sequence 5’-CCA-3’ at their 3’ ends
chain

➢ All tRNA molecules also have a

number of bases modified
chemically by enzyme reactions
with different arrays of
modifications on each tRNA type.
 RNA Characteristics, stability and forms 23

How is an amino acid loaded onto a tRNA?

➢ The correct amino acid is attached to the tRNA by an enzyme called
aminoacyl–tRNA synthetase.

➢ The process is called aminoacylation, or charging, and produces an

aminoacyl–tRNA. The required energy is provided through ATP hydrolysis.

➢ Each enzyme recognizes particular structural features of the tRNA or tRNAs

it aminoacylates.
 RNA Characteristics, stability and forms 24

3
4
 RNA Characteristics, stability and forms 25

How is an amino acid loaded onto a tRNA?

1) the amino acid and ATP bind to the specific aminoacyl–tRNA synthetase
enzyme.

2) The enzyme then catalyzes a reaction in which the ATP is hydrolyzed to

AMP, which joins to the amino acid as AMP to form aminoacyl–AMP.

3) The tRNA molecule binds to the enzyme, which transfers the amino acid
from the aminoacyl–AMP to the tRNA and displaces the AMP.

4) The enzyme then releases the aminoacyl–tRNA molecule.

5) Chemically, the amino acid attaches at the 3’ end of the tRNA by a covalent
linkage between the carboxyl group of the amino acid and the 3’OH or 2’OH
group of the ribose of the adenine nucleotide found at the 3’ end of every
tRNA.
 RNA Characteristics, stability and forms 26

How is an amino acid loaded onto a tRNA?

 RNA Characteristics, stability and forms 27

Ribosomal RNA (rRNA)

➢ Ribosomes are the site of protein synthesis, they bind to mRNA and
facilitate the recruitement of the proper amino acid loaded tRNA.

➢ In both prokaryotes and eukaryotes, ribosomes consist of two

unequally sized subunits—the large and small ribosomal subunits—
each of which consists of a complex between RNA molecules and
proteins.

➢ Each subunit contains one or more rRNA molecules and a large

number of ribosomal proteins.

➢ rRNA is the most abundant RNA in the cell (80%).

➢ In prokaryotes and eukaryotes, the regions of DNA that contain the

genes for rRNA are called ribosomal DNA (rDNA) or rRNA
transcription units.
 RNA Characteristics, stability and forms 28

Ribosomal RNA (rRNA)

 RNA Characteristics, stability and forms 29

Ribosomal RNA (rRNA)

S for sedimentation speed based on size
and shape
 RNA Characteristics, stability and forms 30

Ribosomal RNA (rRNA)

Smaller than the eukaryotic ribosomes and contains only

three rRNAs
 RNA Characteristics, stability and forms 31

Small nuclear RNA (snRNA)

➢ Found in the nucleus of eukaryotic cells.

➢ The average length of snRNA is 150 nucleotides.

➢ Their primary function is in the processing of pre-messenger RNA (hnRNA) in

the nucleus.

➢ snRNA are always associated with a set of specific proteins and these
complexes are referred to as small nuclear ribonucleoproteins (snRNP).

➢ The most common snRNAs are within complexes of the spliceosome

machinery (developed next session).
 RNA Characteristics, stability and forms 32

Small nucleolar RNA (snoRNA)

➢ Small RNA molecules responsible for chemical modifications of others RNAs
non-conventational bases
mainly rRNA and tRNA.

➢ There are 2 mains classes of snoRNAs, one associated with methylation and
the other associated with pseudouridylation. psi nucleotide (pseudouridyl) like slide 22

➢ The pseudouridylation and methylation enhances RNA folding, interaction

with ribosomal proteins and protection against hydrolysis.

➢ They also associate with different proteins to form functional small nucleolar
ribonucleoproteins (snoRNP).
 RNA Characteristics, stability and forms 33

Small regulatory RNA

➢ These small RNA molecules fall into 2 main groups :
• Micro RNA (miRNA)
• Short interfering RNA (siRNA) in a later topic

➢ MicroRNAs are ssRNA regulatory molecules about 21–23 nucleotides (nt)

long that derive from RNA transcripts.

➢ MicroRNAs are encoded by genes in the genomes of all multicellular

eukaryotes, as well as some unicellular ones.

➢ More than 5,000 miRNA genes have been identified among eukaryotes.

➢ Pairing of those miRNAs with their target mRNAs lead to mRNA degradation.
prevent binding of a random small RNA to a
section of a long mRNA molecule (base comp)
➢ MicroRNAs play central roles in controlling gene expression in a variety of
cellular, physiological, and developmental processes.
 RNA Characteristics, stability and forms 34

Long non coding RNAs

➢ Long non-coding RNAs (lncRNAs) are a large and diverse class of
transcribed RNA molecules with a length of more than 200
nucleotides that do not encode proteins.

➢ One of the most studied lncRNAs, XIST (X-inactive specific

transcript) is unusually large (17kb) and plays an important role in
silencing of the inactive X chromosome in females.

➢ XIST RNA coats the X chromosome from which it is transcribed and

recruits a number of enzymes responsible for chromatin remodeling
and epigenetic inhibition of gene expression.