Transcript Slides PPT - The University of Sydney

PHAR2811 Dale’s lecture 3
Genome Structure
COMMONWEALTH OF AUSTRALIA
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Questions from the last lecture
• The role of methotrexate in the inhibition of
thymidylate synthase
• Methotrexate is a competitive inhibitor of
the enzyme that converts dihydrofolate
(DHF) to tetrahydrofolate (THF)
(dihydrofolate reductase, DHFR)
• It binds to the enzyme with ~1000 times
the affinity of DHF...making the inhibition
almost irreversible
Folate conversions
DHF reductase
Dihydrofolate
(DHF)
Tetrahydrofolate,
(THF)
Thymidylate
synthase
Serine
hydroxymethyl
transferase
N5, N10-methylene
THF
H
H2N
N
N
H
H
N
CH 2
O
N
H
R
etc
NADPH + H+
Dihydrofolate, DHF
DHF reductase
TMP
Thymidylate
Synthase
NADP+
H
H2N
N
N
H
H
H
dUMP
Serine Hydroxmethyl
Transferase
O
N
CH 2
H
N
H
H
R
etc
Tetrahydrofolate, THF
H2N
N
N
H
H
H
N
CH 2
CH 2
O
N
R
Serine
Glycine
etc
This
methylene
group is
transferred to
dUMP at C6
Prokaryotes
• The genome of prokaryotes is extremely
efficient.
• There are 4.6 million base pairs in your
average E. coli
• If the average bacterial protein has a
molecular weight of ~40,000 D how many
different proteins does the average E. coli
make?
Prokaryotes
• To do this calculation you need to know:
• The average mol. Wt. of an amino acid
~100
• This means the average protein has 400
amino acids
• Which means 1200 bases + promoter and
terminator sequences  ~1500 bp.
• 4.6 X 106/1500 = ~3000 different proteins.
In humans if the whole genome
was coding
• The genome has 3*109 bp and the
average protein subunit is 50,000
• So 500 aa = 1500 bp = 3000 bp with large
promoter regions which makes the maths
easy
• 3*10 ^9/3*10^3 = 1*10^6 or 1 million
different proteins. We only make about
30,000 different proteins so there is a
discrepancy
Prokaryotes versus Eukaryotes
• Prokaryotes have no room for redundant
sequences.
• Their survival depends on rapid
proliferation when nutrients are available
• Complex multi-cellular eukaryotes depend
for survival on quick responses, adjusting
to changes in the environment.
Prokaryotes versus Eukaryotes
• E. coli can divide every 20 min if
conditions are optimal
• The human cell takes 18 to 24 h to go
through the cell cycle once.
• The human genome only has about 2%
coding regions.
• The gene density is much lower!!
Chromosome Characteristics
• Chromosomes vary in number between
species. The chromosome number is a
combination of the haploid number (n) X
the number of sets. Algae and fungi are
haploid; most animals and plants are
diploid. The number of pairs of
chromosomes in different species’
genomes is bizarre.
What do these life forms have in
common?
Chromosome Characteristics
Species
E. coli
Genome size in # Haploid
Megabases (Mb) chromosomes
4.6
1 circular
yeast
13
cow
human
16
39
3 000
23
alligator
16
carp
52
salamander
90 000
14
Chromosome Characteristics
• Chromosomes vary in size within a
species. Within the human genome there is a
four fold difference in the size of the
chromosomes.
• Centromere: the region of the chromosome
where the spindle fibres attach. Repetitive
satellite DNA is often found around the
centromere.
• Telomere: ends of the chromosome,
containing a distinct repeating sequence, which
enables the ends of the chromosome to
replicate.
Centromere Characteristics
• The relative position of the centromere is
constant, which means that the ratio of the
lengths of the two arms is constant for
each chromosome. This ratio is an
important parameter for chromosome
identification, and also, the ratio of lengths
of the two arms allows classification of
chromosomes into several basic
morphologic types:
Centromere Characteristics
Chromosome Banding
• Chromosomes can be stained with special
dyes which give a consistent and unique
pattern like a bar-code for each
chromosome; so much so that the bands
have been numbered.
• The most common stain used is a Giesma
stain. This stain, when applied after mild
proteolytic treatment (trypsin) gives light
(G-light) and dark (G-dark) bands.
The Human Genome
The ps and qs of chromosomes
• There are 2 arms on the chromosome
denoted p and q
• For most chromosomes the short arm is
the petit or p arm
• The longer arm is the q or queue arm
• Numbering is done from the centromere
along one of the arms
Chromosome Banding
• When viewed at the lowest resolution only a few
bands appear. These are numbered p1, p2, p3
etc counting from the centromere.
• If the stained chromosomes are viewed at higher
resolution many sub-bands are revealed. So the
labelling then goes p11, p12, p13.
• So if your DNA marker may be given a position
on the chromosome with a set of numbers like
17p23. This means the locus is on chromosome
17 on the short p arm in sub-band 23.
Some terms:
• The general material which makes up the
chromosomes is called chromatin This
is composed of DNA and protein.
• Heterochromatin contains DNA which
is more tightly packaged or condensed
and probably is transcriptionally inert.
• Euchromatin contains most active
genes; those actively transcribed.
Chromosome packaging at the
molecular level.
• Each chromosome contains a single
molecule of DNA
• This DNA is wound around small proteins
called histones
• These proteins have lots of lysine and
arginine residues, making them very
positively charged at pH 7 (and high pIs
~12)
Histones
• There are 5 major histone variants: H1,
H2A, H2B, H3 and H4.
• Two molecules each of H2A + H2B + H3 +
H4 make up an octamer which the DNA
wraps around with 1.7 turns.
• This structure is known as a nucleosome.
• Each nucleosome has an H1 associated
and a linker section of DNA, like beads on
a thread.
Histone Octamer
Histones
• The major force holding the association of
histones to DNA is electrostatic.
• To separate the histones from the DNA,
chromatin is treated with high ionic
strength solutions. The high salt reduces
the electrostatic interactions and the
protein dissociates from the DNA.
Higher order Packaging
Figure 11.28
A model for chromosome structure,
human chromosome 4. The 2-nm
DNA helix is wound twice around
histone octamers to form 10-nm
nucleosomes, each of which contains
160 bp (80 per turn). These
nucleosomes are then wound in
solenoid fashion with six
nucleosomes per turn to form a 30nm filament. In this model, the 30-nm
filament forms long DNA loops, each
containing about 60,000 bp, which
are attached at their base to the
nuclear matrix. Eighteen of these
loops are then wound radially around
the circumference of a single turn to
form a miniband unit of a
chromosome. Approximately 106 of
these minibands occur in each
chromatid of human chromosome 4
at mitosis.
The cell cycle
At interphase (G1, S and
G2) the chromosomes
look like a plate of
spaghetti, entangled and
dispersed throughout the
nucleus
At M phase the newly
replicated daughter
chromatids condense
and line up.
Chromosomes at interphase
The role of histones
• Shield the negative charges of the
phosphates
• Allow bending and DNA wrapping
• Restrict access to transcription
• The interaction between the histones and
the DNA is dynamic and non-base
sequence specific
Histone Remodeling
• Influences the DNA accessibility for
transcription
• Can be one of the first events when
switching on a set of genes.
• Nucleosome remodeling complexes
• Nucleosome positioning
• Histone modification
Histone modifications
• Phosphorylation; serine residues
• Methylation, adding –CH3
• Acetylation, lysine residues
Histone modifications
• Phosphorylation; serine residues
• Methylation, adding –CH3
• Acetylation, lysine residues
Acetylation
• Transferring an acetyl group to the amino
side chain of lysine residues
• Histone acetyl Transferases (HATs)
• Histone deacetylases (HDAs)
Lysine
NH
O
C
CH
H2
C
NH
C
O
H2
C
H2
C
H2
C
+NH3
Acetylated Lysine
NH
O
C
CH
O
H2
C
NH
C
O
H2
C
H2
C
H2
C
NH
C
CH 3
What effect would acetylation
have on DNA accessibility?
• It neutralises the positive charge of the
lysine side chain
• The histone will not have as much affinity
for the DNA phosphates (negative)
• The nucleosome packing will be looser
• DNA more accessible for transcription
• Deacetylases will pack it up again!