Molecular Biology II Nucleic Acids 2: Structure and

Function

RNA Processing

RNA undergoes many processing procedures that involve important modifications that relate to its
function.
In both prokaryotes and eukaryotes, tRNA is transcribed as precursor tRNA, then processed.
These transcripts may contain up to 7 tRNA’s.

Pre-tRNA is processed by:

 Cleavage at the 5’ end with RNase P
 Cleavage at the 3’ end with RNase D
RNase P – A small protein + 377 nucleotide RNA molecule. The RNA has enzymic activity so is a
ribozyme.

Post-transcriptional modification of tRNA:

 Intron removal
 Base modification
 CCA addition (eukaryotes only)

H-bonding and stacking interactions lead to the

tertiary structure of tRNA.

Modification of Eukaryotic mRNA:

I) Capping
Chemical modification of the 5’ end to form a
methylated cap
Mitochondrial and chloroplast mRNA is not capped
RNA is unstable – capping helps protect it from a
hostile environment
CEC (Capping Enzyme Complex) is required
Not all RNA is capped

Benefits of capping:
 Regulation of nuclear export
 Prevents degradation by exonucleotides
 Promotes translation
 Promotion of 5’ proximal intron excision

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Molecular Biology II Nucleic Acids 2: Structure and
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Guanylyl transferase adds the terminal G, followed by a methylation reaction (Cap 0)

 Methyl on N7 only
nd
If 2 base is A, it can be methylated (Cap 1)
 N7 and C2 of base ‘X’ methylated
rd
3 base is methylated in ~15% mRNA’s (Cap 2)
 N7 and C2 of bases ‘X’ and ‘Y’ methylated

Some organisms will ‘hyper methylate’, resulting in a different cap structure.

II) Polyadenylation
Addition of 20-200 A’s

The length of an RNA molecule can determine its half-life and its ability to be translated. Half-life’s
can be very short (minutes) so this is important.
1) Cleavage factors bind to specific sequences in the DNA
2) The cleavage factors and stabilising factors bring the 3’ end round in a hairpin shape
3) PolyA polymerase cleaves the 3’ end then synthesis the poly A tail
4) PolyA binding protein (PABP) associates with the tail (70kDa monomer / 10-15 bases).
5) The transcript is now ready for splicing

This process can be used to control gene expression – de-adenylation will inhibit translation of
that transcript.
Histones do not have polyA tails.
PolyA can be useful for mRNA isolation
 Coat beads with many T’s – mRNA will hybridise

III) RNA editing

The coding sequence is occasionally modified:
 CU or UC alterations
 Insertion or deletion of U’s
 Insertion of multiple C or G residues

We don’t know why this occurs – why encode a gene with insertions / deletions that are going to
be modified later? It makes more sense to encode the gene to meet the final requirements. A
waste of time / resources.

Guide RNA’s (gRNA’s) base pair to pre-edited mRNA. They help to edit it in a complex known as
an editosome.

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Molecular Biology II Nucleic Acids 2: Structure and
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Example: Mitochondria of kinetoplastid protozoa (trypanosomes) – hundreds of U’s are removed

to make the mRNA translatable.

IV) Splicing
Involves some kinds of stable RNA that are not mRNA:
 U-RNA (uracil-rich RNA)
 snRNA’s (small-nuclear RNA’s)

Nucleic Acid Labelling:

Can be isotopic (not used so much now)
In the past nucleic acids were labelled with radioactive isotopes ( 32P 33P 35S 3H) and detected using
autoradiography.
 3H – Chromosome in situ hybridisation
32
 P 33P 35S – Used for DNA sequencing

or non-isotopic
Can be labelled directly with a fluorophore or indirectly with a reporter molecule such as biotin.
Visualised using laser detectors, fluorescence, chemiluminescence or colorimetric
assay.

The techniques used for labelling proteins are similar, such as using radioactive C or H.

Methods to label DNA:

I) End labelling (2 methods)

A) Use polynucleotide kinase (PNK) forward or exchange reactions.
PNK catalyses transfer of a -phosphate from ATP to the free 5’-OH of DNA/RNA
Use radioactive ATP (P32ATP)

B) Use Klenow fragment (DNApol) fill-in reaction

Treat DNA sample to leave an ssDNA overhang
Add dNTP’s but select one type (A/T/C/G) to be labelled using a radioactive isotope
The fill-in reaction will occur – all dNTP’s of the type selective will act as radioactive probes.

II) Nick translation

Treat sample of dsDNA with DNase to introduce random ss nicks
E. coli DNApolI will reform the nicks:

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Molecular Biology II Nucleic Acids 2: Structure and
Function

 5’3’ exonuclease activity will remove a nucleotide

 5’3’ polymerase activity will replace the removed nucleotide with a labelled one
Many labelled nucleotides are introduced, the process continues down the DNA for some time
DNA ligase is used to seal the nicks, which have been moved downstream (translated)
The

III) Random priming

dsDNA is denatured using heat to form ssDNA
Random hexanucleotides are added, and the strands are allowed to anneal
The hexanucletotides act as primers.
The Klenow fragment of DNApol is added with a collection of dNTP’s. The nucleotide chosen as
the probe is labelled.
These nucleotides are incorporated onto the DNA fragments.

IV) PCR
A modern technique, but not widely used for labelling
Labelled oligonucleotides can be used to create synthetic duplicates of genes
‘Hot’ nucleotides: 1 – 4 nucleotides are radioactively labelled
High specific activity, expensive  not very useful

Non-isotopic labels for ‘direct labelling’:

 Flourescin
 Rhodamine
 Digoxigenin

Applications of labeled nucleic acids:

Hybridisation techniques:
 Southern
 Northern
 Colony Hybridisation
 Microarray
 In situ hybridisation
DNA Sequencing:
A) Chemical sequencing (Maxam / Gilbert)

B) Dideoxy-chain termination sequencing (Sanger):

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Molecular Biology II Nucleic Acids 2: Structure and
Function

ddNTP’s (dideoxyribonucleotide triphosphates) will cause chain termination, as there is no 3’-OH

for the next nucleotide to bind to. These will be labeled in the reaction.

Similar technique to PCR

Cheap process (£5), sequence length 500-1000bp
1. Four reactions are carried out in vitro:
 ssDNA for sequencing (denature using heat)
 DNA polymerase
 Oligonucleotide primer
 Labeled deoxynucleotides (dATP,dTTP,dGTP,dCTP) – 99%
The reactions differ in that in each tube there will be a different ddNTP – 1%
2. Polymerase will copy the DNA template that has been primed
3. Occasionally, a ddNTP will be incorporated into a growing chain, and will terminate its
elongation
4. This will eventually result in a set of trunctuated DNA fragments at different lengths that are
complimentary to the template DNA
5. Separate these fragments on a urea-containing denaturing polyacrylamide gel at
70C
 Resolution is high enough to separate fragments differing by only 1 base

These chain-terminated products can be visualized by autoradiography (32P/35S-labeled

nucleotides used then expose gel to X-ray film) or fluorescent dyes (each different NTP has a
different fluorophore covalently attached)

This is no longer carried out on gels as they are difficult to automate and are unsuitable for high-
throughput analysis. Instead, they are run through a capillary and detected using a laser.
(See Fairweather 2)

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Molecular Biology II Nucleic Acids 2: Structure and
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Recently, cycle sequencing and PCR have been used to speed up the process and overcome the
secondary structure problems

In automated DNA sequencing, a similar technique is used, involving ddNTP’s, but instead of
radioactive labels, 4 different fluorescent nucleotides are incorporated.
The gel is then run, and a laser / fluorometer system detects the nucleotides as they pass a
detector.
Each base will have a set different wavelength dictated by the fluorophore bound to it. All bases
can be detected in the same lane, no need for a separate lane for each nucleotide as in classical
sanger sequencing.
Data output is peaks of fluorescence at different wavelengths

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Molecular Biology II Nucleic Acids 2: Structure and
Function

Next generation methods:

We now want to know whole genomes

454 and Solexa (brand names) are examples of next gen sequencing techniques
 Easy but uses expensive machinery
 Can sequence entire genome in 1 day
 Powerful methods but not yet widely established

454
1) Obtain a sample of pure dsDNA
2) Fragment dsDNA into smaller pieces
3) Attach adaptors to DNA fragments – Helps amplification of fragments and sequencing
4) Resin beads are added
5) Beads contain primer sequences that are complimentary to parts of the adaptors, allowing
the DNA to attach to the beads. The aim here is to have 1 fragment of DNA / bead.
6) Amplification process in which the fragments are copied many times / bead. Beads are then
filtered to remove any beads that do not contain any DNA.
7) DNA fragments are denatured  ssDNA
8) Each bead is added to its own well
9) Beads containing enzymes are added – polymerase and a primer attach to the DNA
fragment
10) dNTP’s are added in waves

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Molecular Biology II Nucleic Acids 2: Structure and
Function

11) Each time a base is incorporated in replication, some light is given off. The intensity of this
light is proportional to the number of nucleotides of the same type that are incorporated
12) By plotting this sequentially, the sequence of the piece of DNA can be decoded
Find a better description and ensure that I fully understand this…

Solexa
1) Fragment DNA
2) Repair ends and add A’s
3) Ligate adapters and select
4) Attach DNA to flow cell
5) Bridge amplify
6) Generate clusters and anneal primers
7) …
Find proper description and learn.

No cloning
Uses linkers to anchor DNA to slides

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