Download Introduction to Molecular Cell Biology (not tought by SK in 2010)

PDFs of all lectures available from my teaching webpage
Lecture 1:
What is inside the cell?
1. The flow of genetic information: “The Central Dogma of
Molecular Biology”
2. What is inside the cell? - From g
genes to proteins:
p
- Nucleus
- Endoplasmatic reticulum
- Ribosomes
- Golgi complex
- Mitochondria
- Nucleolus
Sergey Kasparov
Room E9
[email protected]
THE CENTRAL DOGMA
Genes (parts of DNA) Intermediates (messenger RNA) Proteins
Prokaryotic versus Eukaryotic cells
Prokaryotic
Eukaryotic
Cells lacking a membranebound nucleus are called
prokaryotes.
From the Greek meaning
“before nuclei”
A eukaryotic cell has the genetic
material organized into a
membrane bound nucleus
Nucleus contains the genetic code.
Cell = nucleus + cytoplasm.
Cytoplasm = cytosol + organelles.
1
e.m.
1.
2.
3.
4.
Nuclear envelope (2 layers of membrane)
Nuclear pores (tightly controlled gates!)
Chromatin (loose strings of DNA, the genetic material)
Nucleolus
Nuclear membrane is a continuation of the
“endomembrane”.
Endoplasmatic
reticulum
What is chromatin?
e.m.
Laemmli
DNA – the molecule which contains the genetic code (which is
a collection of recipes for proteins!).
Between the divisions it needs to be loosely spread to allow
access to its various parts.
2
Nuclear membrane has pores:
Pores are not just “holes”!
Nuclear
membrane
These are specialised micro-channels which are highly selective
and allow traffic of specific molecules from the nucleus and into
the nucleus.
Nuclear pores visualised by
the freeze-fracture technique
Outer
membrane
The genetic instructions
(recipes) stay inside
the nucleus
3
Production of mRNAs is a tightly
controlled process
Signalling
molecules 1. There is a continuous
exchange of information
between the nucleus and
cytoplasm.
2. Specialised proteins
come into nucleus,
nucleus they
bind to DNA to regulate
production of specific
messenger RNAs (mRNAs).
DNA
Messenger 3. Messenger RNAs pass
molecules from nucleus into the
cytoplasm. They encode
proteins which determine
i) cell structure ii) cell
function.
Outside of the nucleus:
the endoplasmatic reticulum
Endoplasmatic reticulum
Rough ER (granular)
Smooth ER (agranular)
4
Two types of Endoplasmatic reticulum
Rough (or granular)
endoplasmatic
reticulum
Organelle which makes the ER “rough”:
The ribosome
Small
subunit
40S
Large
subunit
60S
1. Ribosomes consist of 2
subunits (parts) of
different sizes.
2. Chemically ribosomes are
comprised of a special
type of RNA (rRNA) and a
number of small proteins.
3. They synthesise proteins
according to the sequence
of the messenger
molecules (mRNA) which
arrive from nucleus.
5
Point of Interest:
Ribosomes of bacteria and mammalian cells are
different in their composition. Because of that
antibacterial drugs (antibiotics) may selectively block
protein synthesis
p
y
in bacteria and kill them.
Summary so far
The key points:
1. The messenger (mRNA) molecule arrives from the
nucleus and acts as a template.
2. Subunits of the ribosome “embrace” this template
and act as a docking station for the arriving building
protein (aminoacids)
(
)
blocks of the p
3. The sequence of these blocks is encoded by the
messenger (mRNA)
4. Ribosomes of bacteria and mammalian cells are
different in their composition.
Components of ribosomes are produced in
the nucleus by the structure known as
the nucleolus
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Proteins which need to be secreted out of the
cell are directed from R-ER into the Golgi
apparatus (Golgi complex)
Rough ER (granular)
Smooth ER (agranular)
Smooth (or agranular)
endoplasmatic
reticulum
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Smooth (agranular) ER:
1. Tubular network that does not have
ribosomes attached to it.
2. Functions:
• A. Contains machinery
y for production
p
of
certain molecules (i.e. lipids)
• B. Stores and releases calcium ions, which
control various cell activities, for example
contraction of (cardiac) muscle cells
Mitochondria (singular: mitochondrion)
The key points:
1. Mitochondria are numerous small organelles <
1μM in size. They are the major site of cell
energy production from ingested nutrients.
2. This process involves oxygen consumption and
CO2 formation.
formation It leads to formation of ATP
(adenosine triphosphate) – the fuel of the cell.
3. Cells which utilize large amounts of energy
contain as many as 1000 of them.
4. Have two layers of membrane. The inner layer
forms “cristae” –membrane folds to increase
the inner surface area.
5. May replicate independently of the host cell.
8
The origin of mitochondria:
It is thought that mitochondria have originated
from bacteria which have “learned” to live inside of
their host cells permanently and be useful, rather
than harmful.
Salmonella bacterium
Evolution of cells
Additional interesting facts:
1.
Mitochondria have their own DNA, which replicates independent of the nuclear
DNA
2. Genetic code of the mitochondria is different from the main code of the cell
3. Mitochondria have their own ribosomes on which some of the mitochondrial proteins
are produced. Others are imported from the outside
4. There are genetic disorders which are due to mutations in mitochondrial genes
5. We inherit our mitochondria from mothers because sperms only release their DNA
during fertilisation
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Cytoskeleton
component
Diameter
Building blocks
(protein
monomers)
Examples of function
Microfilament
7 nm
G-Actin
Ubiquitous component of
cytoskeleton,
Contractile protein of
skeletal muscles.
m
.
Microtubule
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Tubulin
Support membrane
protrusions.
Act as intracellular
“railways”.
Separate chromosomes
during cell division.
DNA - RNA - Protein
Nucleus: contains DNA, the source of the genetic code.
It is responsible for storage of the code and its retrieval
(e.g. production of mRNAs for the proteins required by
the cell)
Ribosomes: assemble proteins according to the
instructions relayed by the messenger RNA
Nucleolus – a dense spot
p in the nucleus where the
rRNA required for production of ribosomes is produced
Rough Endoplasmatic Reticulum: provides the
scaffolding for the ribosomes and is involved in protein
maturation
Golgi complex: a system of cisterns involved in protein
sorting and traffic
Mitochondria: supply fuel (ATP) for all cellular
activities, including genome function
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PDFs of all lectures available from my teaching webpage
Lecture 2
THE CENTRAL DOGMA OF
MOLECULAR BIOLOGY:
The Universal Genetic Code
Information Transfer
From DNA-to- mRNA – to - Protein
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States of DNA
Chromosomes – supercoiled
DNA just before cell division
DNA – the molecule which contains the genetic code.
Between the divisions it is loosely spread in the nucleus
which is essential to allow access to its various parts.
Nucleotides: the letters of the genetic code
Components of a nucleotide:
a) A sugar
Found in RNA
Found in DNA
Nucleotides: the letters of the genetic code
Components of a nucleotide:
a) A sugar
b) A residue of phosphoric acid
c) One of the bases
O
-O-P-O
12
H
DNA is built of numerous
nucleotides connected via
phosphoric acid residues
DNA is a linear molecule
and it has a “beginning”
(5’ end with phosphoric
acid residue attached) and
“end” (3’ end with free
sugar OH group).
H
backbone
?
TOO FAR
?
E. Chargaff’ law: the amount
of adenine is always equal to
that of thymine, and the
amount of guanine to that of
cytosine.
A & T and C & G form
complimentary pairs. Thus, for
each sequence of nucleotides
there is a complimentary
sequence.
13
DNA:
the double helix
MOVIE
Why go double stranded –1 ?
Double-stranded conformation has very low
potential energy (e.g. is the most
thermodynamically favourable state).
Why go double stranded –2 ?
Always keep a back-up copy: in case one of the
strings gets damaged
damaged, the other can be used as a template to
restore the information:
14
It is possible to break the two
strings apart by heating the DNA.
This process is often called
“melting”.
However when DNA cools down the
two strings will re-anneal!
Heat
Cool
Points to NOTE:
1.
Sequences with high CG (3 bonds) content are much more
difficult to break apart than those with high AT content (2
bonds). How can we guess the GC content of a DNA sample from
its melting temperature?
2. RNA and DNA may anneal to each other. This is called
hybridisation and is used in some experimental techniques.
3. DNA contains thymine and RNA uracil, but their role are
essentially
ss ti ll id
identical
ti l
4. It may happen that only some parts of the molecules (RNA &
DNA) are complementary to each other. They will anneal to each
other but non-complimentary parts of the molecules will stay
separate.
complimentary
non-complimentary
How can this system be used to
encode proteins???
15
1. Hypothesis of DNA-protein co-linearity:
DNA sequence in some way describes the
sequence of aminoacids
DNA
(a sequence of bases)
PROTEIN
(a sequence of aminoacids)
2. It is known that there are ~20 aminoacids
How many nucleotides need to combine
to encode 20 aminoacids???
There are 4 nucleotides available
41 = 4 amino acids
42 = 16 amino acids
43 = 64 amino acids
3-nucleotides are needed to make one “codon”
DNA
PROTEIN
Mutation –1 : ONE nucleotide added
PROTEIN
Mutation –2 : TWO nucleotides added
PROTEIN
Mutation –3 : THREE nucleotides added
PROTEIN
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Triplets of nucleotides in DNA encode
individual aminoacids
T
in DNA
T in DNA
T
in DNA
T in DNA
T in DNA
T in DNA
Essential features of the code:
1. Linearity
2. Redundancy
3. “Wobble”: the 3rd base is the least
important
3 S
3.
Specialised
i li d codons:
d
- Initiation codon (ATG – methionine)
- Stop codons which do not encode any amino
acid -!!!!!!!!!
4. Genetic code is UNIVERSAL (e.g. nearly the
same in plants, bacteria, humans, insects
etc)
17
Genetic code may be used for
prediction of possible proteins
based on DNA sequence analysis
etc …
approx. 5000 bp
ATG
NOTE:
Only one DNA strand contains the information about the sequence
of the protein. This is called the “sense” strand. The other strand
(“antisense”) is just its mirror image. In order to reveal protein
sequence one needs to know the sequence of the “sense” strand).
TA
Computers automatically detect all possible “open
reading frames”. Usually it is possible to guess which
one is the right one based on its length.
Example: protein which might be encoded by this RNA has a
molecular mass which corresponds to ~ 1700 amino acids. The gene
has to have an ORF of ~5100 base pairs.
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Mutations: accidental alterations of the code
No shift in
the reading
frame: only a
single codon
altered
Possible results of a “substitution” mutation:
Original:
AGG TAG CGA CTT
Arg Trp Arg Leu
AGG TAT CGA CTT
Arg Cys
Arg Leu
AGG TAA CGA CTT
Arg
Consequences of missense mutations at the level of protein will depend
on the nature of substitution (i.e. which amino acid replaces which).
Substitutions may be “silent” (especially if it is the 3rd base in a
codon).
Mutations: accidental alterations of the code
Shift in the reading
frame:
Everything past this
point will be
different!
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Gene expression
DNA – mRNA - PROTEIN
Each cell is producing its
individual set of proteins.
Signalling
molecules
Specialised proteins
(transcription
bind to DNA to regulate
production
d
i
of
f specific
ifi messenger
RNA (mRNA).
factors)
Genes which actively generate
mRNA are “EXPRESSED”.
Messenger
molecules
DNA
Summary
9 The information in DNA is stored in codons (triplets
of nucleotides) and is read linearly. A shift in
reading frame will completely change the whole
message.
9 From the sequence one may make guesses about
proteins which it might encode
9 Mutations are “unauthorised”
unauthorised alterations of the
code. They do not always have visible consequences
and may be beneficial or lethal.
9 Knock-out animals bare artificially introduced
inactivating mutations in genes which scientists are
interested in.
9 Genes may be dormant or “expressed” – this is when
they start producing numerous molecules of mRNA
and protein.
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PDFs of all lectures available from my teaching webpage
Lecture 3
The road from information to
function:
1 Transcription
Tr nscripti n
2 RNA processing
3 Translation
4 Posttranslational processing of
proteins
Mendel’s inheritable
factors – genes.
Definition of a gene?
-a unit of heredity, it
controls a single
inheritable
characteristic
characteristic.
- a DNA sequence
which encodes for a
single polypeptide chain
or a single mRNA.
Essential features of the code:
Make sure you have this information!
1. Each “word” consists of 3 “letters” and is called a “codon”
2. There are no gaps between the “words”. Therefore if the
“frame” of reading is shifted the whole sense changes
3. The code is linear: the sequence of nucleotides in the DNA
is read in one direction from the beginning (5’ end) to the
end (3’ end)
4. The code is redundant (e.g several words mean the same
thi )
thing)
5. It “wobbles”: the 3rd base (3rd letter in each word) is the
least important
6. It has specialised codons :
- The initiation codon (ATG – methionine)
- Stop codons which do not encode any amino acid
7. Genetic code is UNIVERSAL (e.g. nearly the same in plants,
bacteria, humans, insects etc)
8. In DNA the letters of the code are: ACTG. In RNA the
letters of the code are ACUG
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ATG
TA
BASIC FLOW OF INFORMATION IN A CELL:
DNA – RNA – PROTEIN
Structure of a gene
PROTEIN
1. Exons
2. Introns
Only a few percent of human genome
are currently thought to be exons!
The genome contains cryptic messages!
ELE XGTIEN PHA EMRTI NT
ELE XGTIEN PHA EMRTI NT
ELEPHANT
PROTEIN
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Primary RNA transcript
1
RNA
SPLICING
2
3
Mature RNA
Contains sequences from the exons
ONLY!
How does the transcription machinery work?
5’ end of the
“sense”
strand
RNA polymerase
gene
In order for transcription to begin DNA must
unwind and form a “fork” so that RNA polymerase
can get attached to it.
Promoter sequences
5’
Transcription
factors
RNA polymeraseII
Coding part
of the gene
1. Promoter sequences are located upstream of
the gene
2. Specific proteins known as transcription
factors can bind to these sequences and
facilitate transcription
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regulatory gene(s)
RNA
polymerase
PROMOTER
regulatory gene(s)
the gene
Would you like to make an entry into your handouts?
RNA
polymerase
PROMOTER
structural gene
Mature mRNA
1
5’ end
2
Information
Information
3
Poly-A tail
3’ end
Think:
What would happen if this mRNA was hybridised
with its parent genomic DNA?
24
Important:
The code in the mature mRNA is
identical to that in the exons of the
sense (coding) strand of DNA.
Thus, mRNA can deliver correct
information from the gene to the
ribosome where protein synthesis will
occur.
Translation:
The process of synthesis of proteins
according to the information delivered by
mRNA
Amino acids may have very different properties:
1.
2.
3.
4.
Size
Charge
Solubility in water and lipids
Ability to form bonds with other amino acids and various
molecules
25
Key points:
mRNA arrives with a “cap” and a poly A tail
The ribosome scans the mRNA from 5’ end
to find the first methionine codon
Translation is then initiated and more and
more amino acids arrive attached to the tRNAs
When the ribosome reaches a stop codon,
translation is terminated and the new peptide
chain dissociates from the ribosome
The process will repeat until mRNA looses its
poly-A tail and gets degraded
Posttranslational Protein Modifications
(Maturation)
1. Removal of certain parts of the protein molecule
2. Addition of some residues (sugars & lipids) to specific amino
acids in the new protein
3. Formation of intra-protein bonds
Traffic of new proteins from the ER to the Golgi complex for
further processing and (in some cases) secretion from the cell
What have we done…
1. Revised the structure of the cell and its basic
machinery for storage of genetic information and
protein production
2. Got familiar with the “Central Dogma” of
molecular biology
3. Looked at the Universal Genetic Code and its
li ti tto th
the genomic
i d
data,
t such
h as DNA and
d
application
RNA sequences
4. Discovered how the information stored in DNA is
transferred to RNA and then used to determine
the structure and functional properties of
proteins
5. … and how proteins “mature”…
26
PDFs of all lectures available from my teaching webpage
Lecture 4
Wonders of Molecular Biology
Nuts and bolts of DNA manipulation
p
Transferring genes from jellyfish
and coral into brain nerve cells
“Wonders of Molecular Biology”
27
Why manipulate the DNA?
Because we want to understand how the system
works in health and disease
- Find an abnormality responsible for a disease
- Generate an animal model to study a disease
- Search for the drug target(s) or screen the
chemical molecules for their ability to bind to this
target
- Establish new research methods with better
resolution and relevance to human diseases
Tweak it
Get it
Put it
back in
Rat brain neurones made fluorescent with gene transfer
C
A
pin
copper wire
carbon adhesive
cyanacrylate
y
y
adhesive
glass
carbon fiber
silicone elastomer
small clear vesicles
neuronal dense core vesicles
‘chromaffin granule-like’ large vesicles
28
Two different types of brain cells made to glow
green and red using gene transfer
Green Fluorescent Protein
Red Fluorescent Protein
Getting the Gene out of the Cell
Mature RNA
DNA made by “reverse transcription”
Reverse transcriptase allows
generation of the DNA version of
the mature RNA
The moral: do not believe dogmas!
29
The basic toolkit for DNA manipulation:
1. DNA which encodes the gene (must be
sequenced for further analysis)
2. Restriction endonucleases (special
enzymes) to cut and other enzymes to
glue bits of DNA together
g
g
3. Plasmids to proliferate DNA
4. PCR (to generate numerous copies of the sequences
we know, to introduce mutations and “sticky ends”)
Restriction Endonucleases
DNA sequence
etc …
approx. 5000 bp
30
Make a map with restriction sites
EcoR1
EcoR1
Plasmids:
Small self-replicating DNA molecules which live
in bacteria
31
DNA of interest
EcoR1
5’
G A T T C
5’
Plasmid DNA
EcoR1
C T A A G
EcoR1
C T A A G
EcoR1
G
AT T C
C T A A
G
“Sticky ends” find each other!
DNA ligase
G AT T C
C T A A G
MOVIE PLASMID CLONING
32
Putting the Gene Back into
the Cell
Our gene delivery vehicles
Adenoviral vectors
E1
Early ‘E’ genes
0
ITR
E3 E4
E2
20
40
L1 L2
L3
60
80
L4
100
36 kb genome
ITR
Late ‘L’ genes
L5
Late transcription
TRANSGENE
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WHAT IS IT WE NEED TO DO TO
“EXPRESS A GENE?”
PROMOTER
THE GENE
POLY-A signal
3’ end
5’ end
An example of an
expression cassette
m RNA
TRANSCRIPTION
FACTOR(S)
How can we make these vectors selective for
certain types of cells?
Promoter sequences
5’
Transcription
factors
RNA polymeraseII
Coding part
of the gene
• Transcription factors bind to SPECIFIC
SEQUENCES in gene promoters and facilitate
transcription…
Each cell is producing its individual
set of proteins because…
each cell has a specific blend of
active transcription factors
A
B
THE GENE
POLY-A tail
3’ end
5’ end
PROMOTER
Binding sites for the
TRANSCRIPTION
FACTOR(S)
m RNA
34
Two different types of brain cells made to glow
green and red using gene transfer
Gene manipulation in the brain reduces
blood pressure in an animal model of
hypertension
(spontaneously hypertensive rat)
EGFP in SHR
160
Systolic
Blood
140
pressure
p<0.05
120
100
eNOSi in SHR
eNOSi in WKY
0
7
14
Waki et al Hypertension 2006
Summary
1. DNA fragments may be cut out of their original
context and re-cloned into new DNA molecules
such as plasmids.
2. Foreign genes may be expressed in various cells.
An expression cassette may be placed into a
viral vector to deliver this gene to
differentiated cells in the body.
body
3. It is possible to target genes to specific cell
types in the body (selected brain cells, cardiac
muscle cells, blood cells etc) using cell-specific
promoter sequences.
4. Viruses may be genetically modified and used as
gene delivery tools. This is one of the
approaches to human gene therapy.
35
Can you suggest a caption
for this picture?
SEE YOU LATER, ALLIGATOR!!!
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