Download DNA - Laboratory of Theory of Biopolymers

Struktura polimerów i
biopolimerów (3)
Andrzej Koliński
Pracownia Teorii Biopolimerów
Wydział Chemii, Uniwersytet Warszawski
[email protected]
http://www.biocomp.chem.uw.edu.pl
Łańcuch idealny (swobodnie związany): <R2> ~ n
<S2>= <R2> /6
oraz
Łańcuch rzeczywisty (atermiczny): <R2> ~ n6/5
Współczynnik ekspansji: α2 = <R2>/<R2o>
α5−α3 = const. (1−Q/T) n1/2
(Teoria Flory,ego)
Skrajne wartości T 
R
2 1/ 2
= aN
R
3/ 5
T = Q:
R
2 1/ 2
= aN1 / 2
2 1/ 2
 aN 1/ 3
Polymer solutions and melts
Polymer solutions and melts
polymer melts
many-chain problem
1 23
tagged chain
m
“tagged chain“ in a “matrix“
rm
0
n
rn
N-1
N
Polymer entanglements
De Gennes „reptation” theory
De Gennes „reptation” theory
• Originally proposed for
polymer diffusion in crosslinked gels
• Then extend on polymer
solutions
• Explains some experimental
facts (3.0 and -2.0 exponents
for the viscosity and diffusion
chain length dependences)
• Wrong exponent for viscosity
(3.0 versus experimental 3.43.6)
• Does not differentiate between
the critical points for viscosity
and diffusion
• Completely wrong for cyclic
polymers
The problem can be resolved by properly designed computer
simulations
SIMULATIONS OF MULTIPLE
CHAIN SYSTEMS – FAILURE
OF THE REPTATION MODEL
- Coarse-grained polymer
model
- Many chains in a periodic box
- Monte Carlo dynamics
- The snapshots of single
chains shown for clarity
- Lateral mode of motion
dominates
- The measured dynamic
properties agree with
experiment
SIMULATIONS OF MULTIPLE
CHAIN SYSTEMS – FAILURE
OF THE REPTATION MODEL
- Coarse-grained polymer
model
- Many chains in a periodic box
- Monte Carlo dynamics
- The snapshots of single
chains shown for clarity
- Lateral mode of motion
dominates
- The measured dynamic
properties agree with
experiment
A. Kolinski, J. Skolnick and R. Yaris, "Monte Carlo Studies on the Long Time Dynamic Properties of
Dense Cubic Lattice Multichain Systems. I. The Homopolymeric Melt", J. Chem. Phys. 86:71647174 (1987).
A. Kolinski, J. Skolnick and R. Yaris, "Monte Carlo Studies on the Long Time Dynamic Properties of
Dense Cubic Lattice Multichain Systems. II. Probe Polymer in a Matrix of Different Degrees of
Polymerization", J. Chem. Phys. 86:7174-7180 (1987).
Polymer motion in concentrated solutions and melts
1. Rouse-like motion of segments
2. Some segments move slower, due to the dragging of other chains
Stress
Polymers: Deformation
I
II III IV
Strain
I. Chain unfolding, unwinding, unwrapping or uncoupling (low energy)
II. Chain sliding (low energy)
III. Bond stretching, side group ordering (high energy)
IV. Bond breaking (high energy)
Stress
Polymers: Deformation
Ceramics
Metals
Polymers
Strain
•Lower elastic modulus, yield and ultimate properties
•Greater post-yield deformability
•Greater failure strain
(Strain-Stress
> odkształcenie-naprężenie)
Polymers: Viscoelasticity
• Dependency of stress-strain behavior on time
and loading rate
• Due to mobility of chains with each other
• Crosslinking may affect viscoelastic response
Stress
increasing loading
rate
Strain
Polymers: Thermal Properties
decreasing temperature
or increasing crystallinity
log(Modulus)
Stress
• In the liquid/melt state enough thermal energy
for random motion (Brownian motion) of chains
• Motions decrease as the melt is cooled
• Motion ceases at “glass transition temperature”
• Polymer hard and glassy below Tg, rubbery
above Tg
Tg
Tm
semicrystalline
crosslinked
linear amorphous
Strain
Temperature
Viscoelastic Deformation
Glassy Materials and Tg
Rigid Brittle Solid below Tg
Viscous Deformable liquid
above Tg
F
A

d
v
dx
η is the viscosity
of the fluid

Q 

 o  exp 

 R T 
Viscoelastic Modulus versus Temperature
Fracture of Polymers
•Thermoset and Thermoplastic materials below Tg behave as brittle solids
and fail by cracking. The cracks are sharp.
•Crystalline Thermoplastic Resins above Tg yield and undergo ductile failure.
•Noncrystalline Thermoplastic resins above Tg undergo Crazing and
Cracking.
• Crazing - Orientation of polymer chains across the opening of a “cracklike” feature. The work of crazing is 1000 times larger than the surface work
to create a crack.
•Cracking then occurs down the middle of the craze.
Craze Formation
Crack Propagation Through Crazed Area
Cracking Example in Polymers
Polymers and Biopolymers
Molecular Biology and Structure of
Biopolymers




nature of the genetic code
maintenance of genes through DNA replication
transcription of information from DNA to mRNA
translation of mRNA into protein.
DNA
mRNA
protein
J. Skolnick, J. Fetrow and A. Kolinski, "Structural genomics and its importance
for gene function analysis", Nature Biotechnology, 18:283-287 (2000)
Problem „drugiej części” kodu genetycznego
Sequences and structures
KNOWN STRUCTURES
~ 100,000
FR
NEW FOLDS ?
COMPARATIVE
MODELING
KNOWN SEQUENCES
~ 50,000,000
Ścieżka zwijania CI2
S. Kmiecik & A. Kolinski. „Characterization of Protein Folding Pathways by Reducedspace Modeling.” Proceedings of the National Academy of Sciences of the USA,
104(30):12330-5, 2007
Literatura
J. Setubal, J. Meidanis: „Introduction to
computational molecular biology”
P. C. Turner, et al.: „Instatnt notes in molecular
biology”
C. Branden, J. Tooze: „Introduction to protein
structure”
Scheme of a cell:
an introduction
Nucleic acids (DNA and RNA)
• Form the genetic material of all living
organisms.
• Found mainly in the nucleus of a cell
(hence “nucleic”)
• Contain phosphoric acid as a component
(hence “acid”)
• They are made up of nucleotides.
Nucleotides
• A nucleotide has 3 components
– Sugar (ribose in RNA, deoxyribose in DNA)
– Phosphoric acid
– Nitrogen base
•
•
•
•
Adenine (A)
Guanine (G)
Cytosine (C)
Thymine (T) or Uracil (U)
Purines & Pyrimidines
Nucleic acids are polymers of nucleotides.
Each nucleotide includes a base that is either
 a purine (adenine or guanine), or
 a pyrimidine (cytosine, uracil, or thymine).
Nucleoside bases found in RNA:
O
NH2
N
N
N
N
H
adenine (A)
N
HN
H2N
N
guanine (G)
O
NH2
N
H
NH
N
N
H
cytosine (C)
O
N
H
uracil (U)
O
Some nucleic acids contain modified bases. Examples:
Nucleoside bases found in RNA:
O
NH2
N
N
N
N
H
N
HN
H2N
adenine (A)
O
NH2
N
H
N
NH
N
N
H
guanine (G)
N
H
O
cytosine (C)
O
uracil (U)
Examples of modified bases found in tRNA:
O
NH2
CH3
NH2
+
H3C
N
+N
N
N
H
N
HN
H2N
N
N
H
O
+
CH3
N
N
H
HN
O
NH
N
H
O
1-methyladenine (m1A) 7-methylguanine (m7G) 3-methylcytosine (m3C) pseudouracil ()
In a nucleotide, e.g., adenosine monophosphate
(AMP), the base is bonded to a ribose sugar, which
has a phosphate in ester linkage to the 5' hydroxyl.
NH2
NH2
N
N
N
adenine
N
N
N
H
2
O 3P
HO
5' CH2
ribose
adenine
H
O
3'
OH
H
OH
2'
adenosine
N
N
O
CH2
H 1'
H
N
N
N
N
4'
NH2
H
O
H
H
OH
H
OH
adenosine monophosphate (AMP)
Nucleic acids
have a backbone
of alternating Pi &
ribose moieties.
Phosphodiester
linkages form as
the 5' phosphate of
one nucleotide
forms an ester link
with the 3' OH of
the adjacent
nucleotide.
A short stretch of
RNA is shown.
NH2
adenine
N
N
5' end O

O P

NH2
N
N
cytosine
5'
O
CH2
4'
O
H

O
H 1'
H
ribose
O
O
O
5'
CH2
O
O
H
H
H

O
P
O
ribose
H
OH
3'
O
nucleic acid
N
H
OH
2'
3'
P
N
O
O
(etc)
3' end
H
cytosine (C)
N
N
O
O
H
guanine (G)
N
N
N
G
H
N
N
H
C
NH
H
H
G  C base
pair in tRNA
Hydrogen bonds link 2 complementary nucleotide bases on
separate nucleic acid strands, or on complementary portions
of the same strand.
Conventional base pairs: A & U (or T); C & G.
In the diagram at left, H-bonds are in red. Bond lengths are
inexact. The image at right is based on X-ray crystallography
of tRNAGln. H atoms are not shown.
Nucleotides
Nitrogenous
Base
Phosphate
Group
Sugar
Nitrogenous
Base
Phosphate
Group
Sugar
DNA
RNA
A
G
A
A=T
G=C
G
C
C
G
G
A
A
C
TU
C
T
U
G
G
Watson and Crick
DNA
Proteins
• Composed of a chain of amino acids.
20 possible groups
R
|
H2N--C--COOH
|
H
Fig. 6.1
Fig. 6.2. Acidic and basic amino acids.
Fig. 6.2. Neutral, non-polar (hydrophobic) amino acids.
Fig. 6.2. Neutral, polar (hydrophilic) amino acids.
Proteins
R
|
H2N--C--COOH
|
H
R
|
H2N--C--COOH
|
H
Amino acids are joined to form unbranched polypeptides by a
peptide bond.

Covalent bond between the carboxyl group of one amino acid
and amino group of the next amino acid.
Fig. 6.3
N-terminus
C-terminus
5’ (DNA)
3’ (DNA)
Dipeptide
This is a peptide bond
R O
R
| II
|
H2N--C--C--NH--C--COOH
|
|
H
H
Peptide bond
Protein structure
• Linear sequence of amino acids folds to
form a complex 3-D structure.
• The structure of a protein is intimately
connected to its function.
Proteins show four hierarchical levels of structural organization:
1.
Primary structure = amino acid sequence

2.
3.
Secondary structure = folding and twisting of a single
polypeptide chain.

Result of weak H-bonds and electrostatic interactions

e.g.,
-helix (coiled) and -pleated sheet (zig-zag).
Tertiary structure = three dimensional shape (or
conformation) of a polypeptide chain.

4.
Determined by the genetic code of the mRNA.
Function of R groups contained in the polypeptide.
Quaternary structure = association between polypeptides in
multi-subunit proteins (e.g., hemoglobin).

Occurs only with two or more polypeptides.
Fig. 6.4
DNA in action
• Questions about DNA as the carrier of
genetic information:
– How is the information stored in DNA?
– How is the stored information used ?
The need for an intermediary
• Fact 1 : Ribosomes are the sites of protein
synthesis.
• Fact 2 : Ribosomes are found in the
cytoplasm.
2 types of cells: Prokaryotes v.s.Eukaryotes
The need for an intermediary
The need for an intermediary
• Fact 1 : Ribosomes are the sites of protein
synthesis.
• Fact 2 : Ribosomes are found in the
cytoplasm.
• Question : How does information ‘flow’
from DNA to protein?
The Intermediary
• Ribonucleic acid (RNA) is the
“messenger”.
• The “messenger RNA” (mRNA) can be
synthesized on a DNA template.
• Information is copied (transcribed) from
one strand of DNA to mRNA.
(TRANSCRIPTION)
Next question…
• How do I interpret the information carried
by mRNA?
• Think of the sequence as a sequence of
“triplets”.
• Think of AUGCCGGGAGUAUAG as AUGCCG-GGA-GUA-UAG.
• Each triplet (codon) maps to an amino
acid.
The Genetic Code
• f : codon  amino acid
• 1968 Nobel Prize in medicine – Nirenberg
and Khorana
• Important – The genetic code is universal!
• It is also redundant / degenerate.
How was the genetic code deciphered?
1.
Cell-free , protein synthesizing machinery isolated from E. coli.
(ribosomes, tRNAs, protein factors, radio-labeled amino acids).
Synthetic mRNA containing only one type of base:
UUU = Phe, CCC = Pro, AAA = Lys, GGG = ?
2.
Synthetic copolymers (CCC, CCA, CAC, ACC, CAA, ACA, AAC, AAA):
Pro, Lys (already defined) + Asp, Glu, His, & Thr
Proportion (%AC) varied to determine exactly which codon
specified which amino acid.
3.
Synthetic polynucleotides of known composition:
UCU CUC UCU CUC  Ser Leu Ser Leu
1968: Robert Holley (Cornell), H. G. Khorana (Wisconsin-Madison),
and Marshal Nirenberg (NIH).
The Genetic Code
Fig. 6.8
Translation
• The sequence of codons is translated to a
sequence of amino acids.
• Transfer RNA (tRNA) – a different type of RNA –
matches amino acids to codons in mRNA.
– Freely float in the cytoplasm.
– Every amino acid has its own type of tRNA that binds to
it alone.
• Anti-codon – codon binding crucial.
• Show animation
tRNA
tRNA
tRNA
tRNA
3d structure
The gene and the genome
• A sequence of nucleotides on the DNA
that encodes a polypeptide is called a
gene.
• Genome = Set of all genes in the organism
+ junk stuff (the entire DNA content).
More complexity
• The RNA message is sometimes
“edited”.
• Exons are nucleotide segments whose
codons will be expressed.
• Introns are intervening segments
(genetic gibberish) that are snipped out.
• Exons are spliced together to form
mRNA.
Splicing
frgjjthissentencehjfmkcontainsjunkelm
thissentencecontainsjunk
Central Dogma of Molecular
Biology
DNA  RNA  Protein  Phenotype
•
• Transcription : DNA  RNA
• Translation : RNA  Protein
Central dogma
ZOOM
IN
tRNA
transcription
DNA
rRNA
snRNA
translation
mRNA
POLYPEPTIDE
Information flow
Transcription – key steps
DNA
• Initiation
• Elongation
• Termination
Transcription – key steps
DNA
• Initiation
• Elongation
• Termination
Transcription – key steps
DNA
• Initiation
• Elongation
• Termination
DNA
+
RNA
Promoters
• Promoters are sequences in the DNA just
upstream of transcripts that define the sites of
initiation.
Promoter
5’
3’
• The role of the promoter is to attract RNA
polymerase to the correct start site so
transcription can be initiated.
Step 2-Initiation-requirements:
1.
2.
3.
4.
5.
6.
mRNA
Ribosome
Initiator tRNA (fMet tRNA in prokaryotes)
3 Initiation factors (IF1, IF2, IF3)
Mg2+
GTP (guanosine triphosphate)
Fig. 6.17
Fig. 6.18
Genes can be switched on and
off
• In an adult multicellular organism, there is
a wide variety of cell types seen in the
adult. eg, muscle, nerve and blood cells.
• The different cell types contain the same
DNA though.
• This differentiation arises because
different cell types express different
genes.
Regulation of genes
• What turns genes on and off?
• When is a gene turned on or off?
• Where (in which cells) is a gene turned
on?
• How many copies of the gene product are
produced?
Regulatory sequences
• These are binding sites for proteins, often
short stretches of DNA (~25 nucleotides).
• Inexactly repeating patterns (“motifs”).
• Motifs stand out as highly conserved
regions in a multiple sequence alignment.
Regulatory sequences