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Biochemistry, part 2
Course outline
1
Introduction
2
Theoretical background
Biochemistry/molecular biology
3
Theoretical background
computer science
4
History of the field
5
Splicing systems
6
P systems
7
Hairpins
8
Micro technology introductions
Microreactors / Chips
9
Microchips and fluidics
10
Self assembly
11
Regulatory networks
12
Molecular motors
13
DNA nanowires
14
Protein computers
15
DNA computing - summery
DNA folding
DNA folding

Many
DNA
molecules
are
circular
(e.g.,
bacterial chromosomes, all plasmid DNA).
Circular DNA can form supercoils. Human
chromosome contains 3x109 basepairs and are
wrapped around proteins to form nucleosomes.
Nucleosomes
are
packed
tightly
to
form
helical
filament,
a
structure
called
chromotin.

RNA are much shorter but more diverse
molecules.
They
can
form
various
three
dimensional structures.
Tertial structure in DNA

Supercoils refer to the DNA structure in
which double-stranded circular DNA twists
around each other. Supercoiled DNA contrasts
relaxed DNA;

In DNA replication, the two strands of DNA
have to be separated, which leads either to
overwinding of surrounding regions of DNA or
to supercoiling;

A
specialized
set
of
enzymes
(gyrase,
topoisomerases)
is
present
to
introduce
supercoils that favor strand separation;

The
degree
of
supercoils
quantitatively described.
can
be
Varieties of supercoiled DNA
Linking number

The linking number L of DNA, a topological
property,
determines
the
degree
of
supercoiling;

The linking number defines the number of
times a strand of DNA winds in the righthanded direction around the helix axis when
the axis is constrained to lie in a plane;

If both strands are covalently intact, the
linking number cannot change;

For instance, in a circular DNA of 5400
basepairs, the linking number is 5400/10=540,
where 10 is the base-pair per turn for type B
DNA.
The twist and writhe

Twist T is a measure of the helical winding of the
DNA strands around each other. Given that DNA prefers
to form B-type helix, the preferred twist = number of
basepair/10;

Writhe W is a measure of the coiling of the axis of
the double helix. A right-handed coil is assigned a
negative number (negative supercoiling) and a lefthanded coil is assigned a positive number (positive
supercoiling).

Topology theory tells us that the sum of T and W
equals to linking number: L=T+W

For example, in the circular DNA of 5400 basepairs,
the linking number is 5400/10=540

If no supercoiling, then W=0, T=L=540;

If positive supercoiling, W=+20, T=L-W=520;
The relation between L, T and W
Positive supercoiling
The relation between L, T and W
Negative supercoiling
L, T and W calculation

A relaxed circular, double stranded DNA (1600
bps) is in a solution where conditions favor
10 bps per turn. What are the L, T, and W?

During replication, part of this DNA unwinds
(200 bps) while the rest of the DNA still
favor 10 bps per turn. What are the new L, T,
and W?
1600 bps
L=1600/10=160
W=0 (relaxed)
T=L-W =160
1400 bps
200 bps
L=160
T=(1600-200)/10=140
W=L-T=+20
Nucleosomes

Nucleosomes look like “beads on a string”
under microscope. The beads contain a pair of
four histone proteins, H2A, H2B, H3, and H4
(octamer). The string is double stranded DNA;

The surface of the octamer contain features
that guide the course of DNA such that DNA
can wrap 1.65 turns around in a left-handed
conformation. H1 proteins serves to seal the
ends of the
nucleosomes.
DNA
and
connects
consecutive
nucleosomes
Organisation of chromosomes
Base pairs
per turn
DNA double helix
Packing
ratio
2 nm
10
1
11 nm
80
6-7
‘Beads on a string’ chromatin form
Organisation of chromosomes
Solenoid (6 nucleosomes per turn)
Base pairs
per turn
Packing
ratio
30 nm
1200
~40
60,000
680
Loops (50 turns per loop)
o.25 μm
Organisation of chromosomes
Miniband (18 loops)
Base pairs
per turn
o.84 μm
Chromosome (stacked minibands)
o.84 μm
1.1 106
Packing
ratio
1.2
104
Organisation of chromosomes
Organisation of chromosomes
proteins
Genetic code







4 possible bases (A, C, G, U)
3 bases in the codon
4 x 4 x 4 = 64 possible codon sequences
Start codon: AUG
Stop codons: UAA, UAG, UGA
61 codons to code for amino acids (AUG as well)
20 amino acids – redundancy in genetic code
Amino acids


building blocks for proteins (20 different)
vary by side chain groups

Hydrophilic amino acids are water soluable
Hydrophobic are not

Linked via a single chemical bond (peptide bond)

Peptide: Short linear chain of amino acids (< 30)
polypeptide: long chain of amino acids (which can be

upwards of 4000 residues long).
20 amino acids






















Glycine (G, GLY)
Alanine (A, ALA)
Valine (V, VAL)
Leucine (L, LEU)
Isoleucine (I, ILE)
Phenylalanine (F, PHE)
Proline (P, PRO)
Serine (S, SER)
Threonine (T, THR)
Cysteine (C, CYS)
Methionine (M, MET)
Tryptophan (W, TRP)
Tyrosine (T, TYR)
Asparagine (N, ASN)
Glutamine (Q, GLN)
Aspartic acid (D, ASP)
Glutamic Acid (E, GLU)
Lysine (K, LYS)
Arginine (R, ARG)
Histidine (H, HIS)
START: AUG
STOP: UAA, UAG, UGA
20 amino acids
20 amino acids
The basic amino acid
Peptide bond
Two amino acids
Removal of water molecule
Peptide bond
Formation of CO-NH
Amino end
Carboxyl end
Peptide bond
Polypeptide
Protein structure
There are four basic levels of structure in protein architecture
Protein structure

Primary–sequence of amino acids constituting the
polypeptide chain

Secondary–local
organization
into
secondary
structures such as  helices and  sheets

Tertiary –three dimensional arrangements of the
amino acids as they react to one another due to
the polarity and resulting interactions between
their side chains

Quaternary–number and relative positions of the
protein subunits
Protein structure
Primary structure: amino acid sequence
Protein structure
Secondary structure: α-helix and β-sheet
Amino end
Carboxyl end
Protein structure
Secondary structure: α-helix and β-sheet
Parallel
Antiparallel
Side view
Side view
Protein structure
Secondary structure: α-helix and β-sheet
Protein structure
Tertiary structure: spatial arrangement of amino residues
Protein structure
Quaternary structure: spatial arrangement of subunits
Protein structure
primary
secondary
tertiary
quaternary
Protein structure
Protein function

Every function in the living cell depends on proteins.

Motion
and
locomotion
of
cells
and
organisms
depends
on
contractile proteins. [Example: Muscles]

The catalysis of all biochemical reactions is done by enzymes,
which contain protein.

The structure of cells, and the extracellular matrix in which they
are embedded, is largely made of protein. [Example: Collagens]

Defence by antibodies.

The
receptors
for
hormones
and
other
signalling
molecules
are
proteins.

The transcription factors that turn genes on and off to guide the
differentiation
of
the
cell
and
its
later
responsiveness
signals reaching it are proteins.

and many more - proteins are truly the physical basis of life.
to
Protein function
Protein function antibody
Protein function enzyme
Gene expression
Gene regulation mechanism
Bacteria express only a subset of their genes at
any given time.

Expression of all genes constitutively in
bacteria would be energetically inefficient.

The genes that are expressed are essential
for dealing with the current environmental
conditions, such as the type of available
food source.
Gene regulation mechanism
Regulation of gene
several levels:
expression
can
occur
at

Transcriptional regulation: no mRNA is made.

Translational regulation: control of whether
or how fast an mRNA is translated.

Post-translational regulation: a protein is
made in an inactive form and later is
activated.
Gene regulation mechanism
Transcriptional control
Translational control
Post-translational control
Lifespan of mRNA
Protein
Onset of transcription
Translation rate
Ribosome
mRNA
DNA
RNA polymerase
Protein activation
(by chemical
modification)
Feedback inhibition
(protein inhibits
transcription of its
own gene)
Escherichia .Coli
Gene regulation mechanism
Operon

A controllable unit of transcription
consisting of a number of structural
genes transcribed together. Contains at
least two distinct regions: the operator
and the promoter.
Gene regulation mechanism
Case study of the
operon in E. coli

regulation
of
the
lactose
E. coli utilizes glucose if it is available,
but can metabolize other sugars if glucose is
absent.
Gene regulation mechanism
Food
source:
Glucose : Lactose
Glucose : Lactose
1:3
Glucose : Lactose
1:1
70
60
50
40
30
20
3:1
29.5
14.0
43.5
26.5
39.0
13.5
10
0
0
1
2
3
4
5 0 1
2
3
4
5
Time (hours)
6 0
1
2
3
4
5
6
7
Second
period of
rapid
growth with
lactose as
food source
Initial
period of
rapid
growth
with
glucose as
food
source
Gene regulation mechanism
Case study of the
operon in E. coli

regulation
of
the
lactose
Genes that encode enzymes needed to break
other sugars down are negatively regulated.
 Example:
enzymes required to metabolize
lactose are only synthesized if glucose is
depleted and lactose is available.
 In
the absence of lactose, transcription
of the genes that encode these enzymes is
repressed. How does this occur?
Gene regulation mechanism
Case study of the regulation of the lactose
operon in E. coli
 All the loci required for lactose metabolism
are grouped together into an operon.

The
lacZ
locus
encodes
-galactosidase
enzyme, which breaks down lactose.

The
lacY
locus
encodes
galactosidase
permease, a transport protein for lactose.

The function of the lacA locus is unknown.

The lacI locus encodes a repressor
blocks transcription of the lac operon.
that
Gene regulation mechanism
Regulatory
function
Cleaves lactose
to glucose and
galactose
Regulatory
protein
Lacl
ß-galactosidase
LacZ
Membrane transport
protein-imports
lactose
Galactosidase
permease
LacY
Section of E. coli
chromosome
lacl
lacZ
Observations about
regulation of lacZ and lacY:
(1) Lacl protein and glucose
shut down transcription of
lacZ and lacY
Glucose
Lactose
E. coli
Galactose
(2) Lactose induces
transcription of lacZ andlacY
lacY
Galactosidase
permease
Chromosome
ß-galactosidase
Gene regulation Lac operon
Lac operon
lacl promoter
lacl
Promoter
Operator
lacZ
lacY
lacA
Gene regulation mechanism
Repression and induction of the lactose operon.

The lac operon is under negative regulation,
i.e. , normally, transcription is repressed.

Glucose represses transcription of the lac
operon.
 Glucose
inhibits cAMP synthesis in the
cells.
 At low cAMP levels, no cAMP is available
to bind CAP.
 Unless CAP is bound to the CAP site in
the promoter, no transcription occurs.
Gene regulation mechanism
When no lactose is present, the repressor
binds to DNA and blocks transcription.
NO TRANSCRIPTION
Functional
repressor
lacl
lacZ
RNA polymerase
blocked
Operator
(binding site
for repressor)
lacY
Gene regulation mechanism
Repressor plus lactose (an inducer)
present. Transcription proceeds.
Lactose
TRANSCRIPTION BEGINS
repressor
lacl
+
mRNA
Permease
galactosidase
lacZ
lacY
Gene regulation mechanism
Operons produce mRNAs that code for
functionally related proteins.
"Polycistronic" mRNA
lacZ
message
RNA polymerase
binds to promoter
lacY
message
lacA
message
lacl
promoter
lacl
Promoter
Operator
lacZ
lacY
lacA
DNA binding sites
DNA binding proteins

Proteins that bind to DNA share similarity in
the structure of their DNA-binding regions.

Many
DNA
binding
proteins,
such
as
lac
repressor, have a helix-turn-helix motif which
fits into the major groove of a DNA molecule
DNA binding proteins
(a)
(b)
(c)
DNA binding proteins
Binding of an inducer to the lac repressor
causes it to release the operator DNA because it
alters the conformation of the helix-turn-helix
motif.
DNA binding proteins
DNA binding proteins
DNA binding proteins
DNA binding proteins
Information about regulation of the expression
of genetic loci may help to combat diseases.

Virulent bacterial strains have genes that
encode the ability to infect and produce
disease.

Knowledge of how the expression of these
genes
is
controlled
and
regulated
may
provide
insights
into
blocking
the
development of the disease.
DNA binding proteins, negative regulation
When tryptophan is absent, transcription occurs.
RNA polymerase
Leader
5 coding loci
Promoter
When tryptophan is present, transcription is blocked.
Tryptophan
Repressor
DNA binding proteins
Ribosomes translates
mRNA rapidly when
tryptophan is abundant,…
…leading to formation
of stem-and-loop
structure that inhibits
RNA polymerase and
terminates transcription.