Epigenetic Marks
• Any stable modification of DNA
• DNA modifications
– almost all methylation on C (5methylcytosine)
– mostly at ‘CpG' dinucleotides
– Some other methylation derivatives
• Histone modifications by adding groups
– Majority on histone H3
– Mostly methyl, acetyl, phosphate groups
– Sometimes ubiquitin!
Nucleosomes and Histone Marks
• DNA wrapped around nucleosomes (8 histones)
– ~142 bases wrapped twice
• Nucleosomes may be consistent (‘well positioned’) from cell to cell
at key regulatory sites
CpG Islands and Promoters
• Although C & G constitute 42% of the human genome, less
than 1% of pairs are CpG
– Less than ¼ of expected frequency of 0.04
• A ‘CpG island’ is a run of “CpG-rich” sequence
– Typically taken as > 2% of pairs
– This definition is not precise (there are others)
• Many CpG islands occur within promoters
• In vertebrates >70% of CpG cytosines methylated
• Methylation mediated by DNA methyltransferases
Image: cellscience / alcat.com
Epigenetic Marks at Promoters
• H3K4me3 typically marks active promoters (and a
few other places)
• H3K27ac typically marks active regulatory sites
including promoters
• H3K27me3 marks repressed promoters
– Typically developmental genes
• DNA methylation dips at silenced promoters
• One histone is kicked out at active promoters
Central Dogma:
DNA to RNA to Protein
Processing /
Translocation
Translation
How to use an mRNA to create a protein?
NOT OBVIOUS – RNA can’t hydrogen bond with the 20
different amino acids in a meaningful way
One four base code must be
translated into another 20 amino acid code
Energy intensive process that requires:
• mRNA
• tRNA
• aminoacyl tRNA synthetases
• ribosomes
tRNAs serve as molecular adaptors
between amino acids and codons
• 75 – 95 NT
• exact sequence varies
• 3’ terminus is the AA
attachment site
• most tRNAs can
recognise >1 codon
• contains both paired
and unpaired regions
Many types, all with same basic structure, each attaching
a specific amino acid and recognizing specific codons
tRNAs contain several
non-standard bases
Also thymine, hypoxanthine,
methylguanine, inosine
• All result from post-transcriptional modification of bases
• Improves tRNA function
• Some (hypoxanthine) involved in codon recognition
tRNAs assume a characteristic
“L” shaped 3D conformation
Tertiary structure stabilised by:
• Non-Watson-Crick base pairings between bases
• Base-sugar phosphate interactions
• Base stacking in base-paired regions
Aminoacyl tRNA synthetases
“charge” tRNAs
Amino acids are first adenylylated
(AMP transferred)
tRNA charging
Second step results in release of AMP and charging of tRNA
by attachment of AA to either 2’ or 3’ OH of tRNA
It is an underappreciated fact that there is an
implicit second “structural” genetic code
• Most tRNA synthetases
interact with several different
“isoaccepting” tRNAs
Although there can be many tRNAs for a particular amino acid,
ONLY ONE aminoacyl tRNA synthetase is responsible for
attaching a particular amino acid to all of the appropriate tRNAs.
The anticodon loop and a 3’ “discriminator” base are
key for recognition and specificity
Aminoacyl tRNA synthetases discriminate
between structurally similar amino acids
Free energy differences of only 2-3 kcal/mol are
sufficient to discriminate 99% of the time,
however the actual error rate is 10x less….
Co-crystal stucture of a tRNA synthetase
with its cognate tRNA
3’ acceptor and discriminator
adjacent to the adenylylated AA
Contact with the anticodon
The “universal” genetic code
Dissimilar
anticodons
can encode the
same AA
Mitochondria and some other organisms use a slightly modified code
Many amino acids have more
than one cognate tRNA
However, there is NOT a separate
tRNA for every possible codon…..
The “Wobble” hypothesis
Non-standard base pairing increases the
codon repertoire of many tRNAs
Inosine promiscuously base pairs
with C, U, or A
G-U base pairing
can also occur
tRNA 3D structure suggests why
wobble is restricted to the third position
The 5’ anticodon position is less hindered
by base stacking interactions
The genetic code explains the outcome
of various types of mutations
• Insertion or deletion
of a base results
in a “frameshift” mutation
• Results in incorporation of
incorrect amino acids
• Frameshifts can also cause
premature termination
Single base pair mutations
• Usually result in a “missense” mutation……
Single base pair mutations
…or, if a stop codon is created, a “nonsense” mutation
Mutations can sometimes be
suppressed by a second mutation
Site of previous base
deletion leading to frameshift
Second mutation
correct the frameshift
This is an example of
intragenic suppression
Suppression can also be intergenic
Mutated gene
Mutated gene and
nonsense suppresor
In intergenic suppression, a mutation in a second
gene compensates for a mutation in the first
Translation can proceed in three
possible reading frames
Open Reading Frames (ORFs) corresponding to possible
peptides occur between predicted start and stop codons
The ribosome is a complex
multisubunit ribonucleoprotein
Note: Eukaryotic ribosome has
60S and 40S subunits,
overall size is 80S
• Over 50 proteins
• Three rRNAs
• Huge 2.5 MDalton complex
• Organised into large and small subunits
The ribosome is a complex
multisubunit ribonucleoprotein
50S subunit
tRNA pocket
30S subunit
The eukaryotic ribosome
Note that S, the Svedburg coefficient, does not
measure mass and is not additive
The prokaryotic ribosome
The ribsomes of eukaryotic organelles are
similar to prokaryotic ribosomes
In prokaryotes, transcription and
translation are coupled
NOT true in eukaryotes – translation occurs in the
cytoplasm, not the nucleus
Overview of the translation cycle
Proteins are synthesised in the
N to C direction
Multiple ribosomes can simultaneously associate
with a single mRNA. Such aggregates are known as
polyribosomes or simply polysomes
Chain growth occurs via the
peptidyl transferase reaction
• This is a coupled reaction – energy ultimately comes
from ATP hydrolysis during tRNA charging
• The nascent polypeptide is always tethered to a tRNA
Not all ORFs get translated!
In prokaryotes, the Shine-Dalgarno sequence, also known
as the Ribosome Binding Site (RBS) interacts with the
16S rRNA to recruit the translational machinery
• Sometimes a second RBS is not required in polycistronic
messages when overlap occurs (e.g. 5’ – AUGA – 3’)
Eukaryotes use different signals
to recruit translation machinery
Eukaryotic ribosomes associate with the 5’ mRNA cap and
“scan” in a 5’-3’ direction for start codons (AUG)
The “Kozak” sequence, if present,
increases the efficiency of translation initiation
The ribosome does not
participate in proofreading
Chemical
reduction
(in vitro)
The ribosome is perfectly happy to incorporate the
WRONG amino acid into a growing peptide chain…
Proofreading happens at the level of the aminoacyl tRNA
synthetases, which have a proofreading pocket.
Study Question 11
Degeneracy and frequency of amino acids
Most common
Leu Gly Ser
Least common
Trp Met His
Study Question 12
Single mutation from AGA
Silent: |
Hydrophilic/
Hydrophilic: |
Study Question 12
Single mutation from AGA
Silent: |
Conservative: |
Hydrophilic/
Hydrophilic: ||
Hydrophilic/
Hydrophobic: |
Study Question 12
Single mutation from AGA
Silent: ||
Conservative: |
Hydrophilic/
Hydrophilic: |||
Hydrophilic/
Hydrophobic: |
Other: |
Study Question 8
Why do introns exist?
AAAAAA...AAA
Splicing
AAAAAA...AAA
Splice boundaries highly conserved
Study Question 8
Why do introns exist?
Protein #1
hormone
responsiveness
Protein #2
protein kinase
DNA binding
chromosomal
rearrangement
Hormone-responsive
protein kinase
DNA-binding
protein
Study Question 8
Why do introns exist?
hormone
responsiveness
protein kinase
DNA binding
AAAAAA...AAA
New protein:
Hormone-responsive
DNA-binding protein
Nucleic Acids complementarity
Study Question 4
Example of palindromic DNA
Study Question 5
Analogy: Translation / Tape recorder