Disclaimer: Out of all the people in the class, it’s sad that I’m the one posting notes
on the web. However, it’s always puzzled me why NONE of you 0t8’s put
notes online, so I guess this is better than nothing.
These notes cover Professor Hampson’s first three lectures of the second
half of the first term. They cover everything Professor Hampson talked
about in class but don’t really cover the textbook too much. I’ve included
some notes from my OAC biology class, as I think they complement Professor
Hampson’s notes very well. I hope these notes help you guys somewhat, but
if they don’t, DON’T BLAME ME =)
How DNA Technology Affects Our Lives
-
production of peptides / proteins that are identical to naturally occurring
substances (ie. Insulin)
production of monoclonal antibodies – increase in specificity of particular
treatment
production of enzymes to treat diseases
safer / faster way to produce vaccines
antisense technology (important technology for drug discovery and development)
PCR (Polymerase Chain Reaction) Technology
Production of receptors
Molecular modeling
Gene therapy
Provide better understanding of life cycle of HIV virus
In 2001, the top biotech drug was Epogen, a medication used in the treatment of
anemia.
Sizes of DNA Molecules
Genome:
The entire DNA of the organism
Size of genome:
Prokaryote: (E. Coli – 4 million base pairs)
Eukaryotes (Fruit Fly – 165 million base pairs)
(Humans – 3 billion base pairs)
However, the 3 billion base pairs found in humans only code for 25 000 genes. To
put this into perspective, some worms have 19 000 genes, and thus, this shows that
the difference in organisms is not determined by the number of genes.
Human Genome Project
-
international effort to sequence all 23 human chromosomes
unfortunately, most of human DNA is just junk DNA!! Thus, one must filter
through the junk.
Watson and Crick’s Contributions to Genetics
-
-
Watson and Crick = scientific team that discovered the structure of DNA
This happened in 1953 with the aid of x-ray diffraction
Discovered that DNA is composed of two alpha-helices that are intertwined
These two alpha-helices run opposite to each other
Bases are on the inside of the helix – they run perpendicular to the long axis
- hydrophobicity keeps bases on the inside
- the inside offers some form of protection
- this also helps save space
Watson and Crick = scientific team that discovered the structure of DNA
Sugars (deoxyribose) is on the outside – hydrophillicity outside the cell makes
it happy =)
Helix requires 10 bases to form a complete turn
Adenosine always pairs up with thyamine (via two hydrogen bonds)
-
Guanine always pairs up with cytosine (via three hydrogen bonds – therefore, it
is more tightly bound)
Sequence of bases = genetic sequence
The only variable part of DNA is the base
DNA is always read from a 5’ (phosphate group) to 3’ (hydroxyl group) direction
(Refer to diagram on next page)
5’
3’
3’
5’
DNA Melting
-
occurs through incrase in temperature / alkali (DNA melts and strands come apart)
Annealing = property that two single strands of DNA (which were together
beforehand) can come together again (via a probe)
Meselson & Stahl Experiment
-
proved that DNA is replicated in a semi-conservative manner
-
Bacteria was cultured on a heavy isotope of nitrogen, 15N
Bacteria incorporated heavy nitrogen into their nucleotides
Bacteria were then transferred to a medium containing 14N (lighter, more common
isotope of nitrogen)
This meant that any new DNA synthesized by the bacteria would contain the lighter
14N
By centrifuging, Meselson and Stahl could distinguish different densities of DNA
The first replication in the 14N medium produced a band of hybrid (15N – 14N) DNA.
This disproved the conservative hypothesis (Parent strand of DNA remains intact,
daughter DNA consisted of two strands of new DNA)
A second replication produced both light and hybrid DNA, a result that eliminated
the dispersive hypothesis (each strand of new DNA contains new and old fragments
of DNA)
However, 1st and 2nd generation DNA patterns both supported semi-conservative
model
DNA Replication
Background info:
-
Arthur Kornberg at Stanford University discovered that enzymes and proteins are
needed for DNA to replicate
DNA replication = high fidelity (that is, its accuracy is uber)
Always goes in 5’ to 3’ direction
Enzymes Involved:
Leading Strand
Priming: Primase
Elongation: DNA Polymerase
Replacement of RNA primer by DNA:
Polymerase
DNA Polymerase:
Lagging Strand
Priming for Okazaki fragment: Primase
Elongation of fragment: DNA Polymerase
Replacement of RNA Polymerase by DNA: DNA
Polymerase
Joining of fragments: Ligase
DNA
An enzyme that catalyzes the elongation of new DNA at a replication
fork by the addition of nucleotides to the existing chain.
Step 1:
-
-
Priming DNA Synthesis
DNA polymerase can add a nucleotide only to an
existing polynucleotide that is already paired
with the complementary strand
- Therefore, DNA Polymerase cannot actually
initiate synthesis of a polynucleotide (they
can only add to an existing chain)
- Thus, a short stretch of RNA serves as the
primer.
- An enzyme called primase joins RNA nucleotides
to make the primer
- Another DNA polymerase later replaces the RNA
nucleotides of the primers with DNA versions
- It’s a bit confusing – basically, RNA serves
as a primer, and DNA Polymerase starts adding
nucleotides to it and elongating the chain.
Later on, another DNA polymerase comes in and
replaces the RNA with DNA
- Only one primer is required for the leading
strand
For the lagging strand, each fragment requires a primer – the primers are
converted to DNA before ligase joins the fragments together
Step 2:
-
Elongating a new DNA strand
DNA polymerase adds proper nucleotides to existing chain (following base pair
rules)
Hampson did not talk about this next part of elongation, so it’s mainly for your
information
-
Nucleoside triphosphates are the source of energy that drives the polymerization
of nucleotides to form new DNA
Nucleoside triphosphates are the same thing as ATP except the sugar component of
Nucleoside triphosphates is deoxyribose (ATP uses ribose)
When a nucleoside triphosphate links to the sugar-phosphate background of a
growing DNA strand, it loses two of its phosphates as a pyrophyosphate molecule
DNA polymerase catalyzes the reaction
Hydrolysis of the bonds between the phosphate groups provides the energy for the
reaction
Okay, back to what Hampson was talking about
The Problem of Antiparallel DNA strands
o
o
o
The two strands of DNA are antiparallel
DNA polymerases add nucleotides to the free 3’
end of a growing DNA strand, NEVER to the 5’ end
Thus, a new DNA strand can elongate only in the
5’ to 3’ direction
Leading Strand
-
-
-
Along one template strand, DNA polymerase
can synthesize a continuous complementary
strand by elongating the DNA in the
mandatory 5’ to 3’ direction
The polymerase simply nestles in the
replication fork (the junction between
the zipped and unzipped part of the
replicating DNA) and moves along the
template strand as the fork progresses
This is the leading strand (no duh)
Lagging Strand
-
To elongate the other new sstrand of DNA, polymerase must work along the template
away from the replication fork
Thus, as the replication “bubble” opens, polymerase works its way away from a
replication fork and synthesizes a short segment of DNA
As the bubble grows, another short segment of the lagging strand can be made in a
similar way
Thus, the lagging strand is first synthesized as a series of segments known as
Okazaki fragments
DNA ligase joins the Okazaki fragments into a single DNA strand
Step 3 – Proofreading
-
A team of enzymes detects and repairs damaged DNA
Repair enzymes can excise the damaged region from the DNA and replace it with
normal DNA segment
Exonuclease is synonymous with proof reading ability of polymerase
Exonuclease site is separate from catalytic site
Probability of DNA migrating to exonuclease site increases if there is an error
in base pairing
DNA DOES NOT ALWAYS GET INTO EXONUCLEASE SITE
Ligase helps seal everything together
Summary of DNA Replication
For Your Information (Hampson did not talk about the following – I just put it in case
Hampson asked a question like this)
The Ends of DNA Molecules Pose a Special Problem
-
For linear DNA, the usual DNA replication
machinery is unable to replicate both ends
A gap is left at the 5’ end of each new
strand because DNA polymerase can only add
nucleotides to a 3’ end
As a result, with each round of replication,
the DNA molecules get shorter – this could
lead to potentially disastrous consequences!
Solution
-
Solved by having expendable, noncoding
sequences called telomeres at ends of
their DNA and the enzyme telomerase in
some of their cells
- Telomeric DNA consists of repeating
six-nucleotide units
- In reference to picture:
1) The enzyme telomerase has a short
molecule of RNA with a sequence that serves
as a template for extending the 3’ end of
the telomere
2) The complementary strand of the telomere
is extended by the usual combined actions of
primase, DNA polymerase and ligase. After
the primer is removed, the result is a
longer telomere with a 3’ end overhang
This ends the section on DNA Replication
From Gene To Protein
One Gene – One Polypeptide
-
general rule of thumb – one gene programs for one specific polypeptide (This is
not always true though because of alternative splicing – I’ll get to that later)
Gene Coding is Degenerate
-
-
multiple codons can code for the same amino acids
61 codons code for amino acids
3 code for termination
this decreases the likelihood of a deleterious effect from happening (if there
were only 20 amino acids to code for each amino acid, that would lead to 44 stop
codons – if a mistake was made, the degenerate code allows for the chance that
the mistake would still lead to the same amino acid (or maybe even a different
one). However, without a degenerate code, a mistake would most likely lead to a
stop codon, preventing the protein from being fully expressed – VERY BAD!
3 types of mutation – addition, subtraction, and substitution. Addition and
subtraction are frame shift mutations, while substitution means a different amino
acid is coded for at one codon – none of the other codons are affected (triplet
stays intact)
RNA
-
RNA has no
3 types of
o rRNA
o tRNA
thiamine (T) – it is now uracil (U)
RNA
– ribosomal RNA (80% of all RNA)
– transfer RNA (15% of all RNA)
-
o mRNA – messenger RNA (5% of all RNA)
Transcription of DNA creates mRNA
In RNA, there is no equal ratio of bases – IT IS SINGLE STRANDED
Transcription:
A Closer Look
Transcription:
- Synthesis of RNA under the direction of DNA
- Both nucleic acids use the same language – info is simply transcribed, or
copied, from one molecule to another
- DNA provides a template for assembling a sequence of RNA nucleotides
- Thus, the resulting RNA molecule (mRNA) is a faithful transcript of the
gene’s protein-binding instructions
Transcription requires: 1)
Template (double or single strand of DNA)
2) Activated precursors (all nucleotide triphosphates – ATP, CTP,
GTP, UTP)
3) Divalent metal ions – Mg2+
-
Enzyme responsible for transcription is RNA polymerase, which moves along a gene
from its promoter to just beyond its terminator
It assembles an RNA molecule with a nucleotide sequence complementary to that of
the gene’s template strand
Regulatory Mechanisms
Promoter:
- A specific nucleotide sequence that binds RNA polymerase and indicates
where to start transcribing RNA
- Transcription always starts here (always located upstream of 5’ end)
Transcription Factors: A regulatory protein that binds to DNA and stimulates
transcription of specific genes
Transcription Initiation Complex:
The completed assembly of transcription factors and
RNA polymerase bound to the promoter
-
The enzyme RNA polymerase transcribes
protein coding genes into pre-mRNA
This enzyme initiates RNA synthesis at
promoters that commonly include TATA box
(The TATA refers to the non-template
strand, sequence typically TATAAAA)
a) Within promoter, TATA box is located
about 25 nucleotides upstream from
transcription start point
b) RNA Polymerase cannot recognize the TATA
box and other landmarks of the promoter
on its own. Another protein, a
transcription factor that recognizes the
TATA box, binds to the DNA before RNA
polymerase can do so
c) Additional transcription factors join
the polymerase on the DNA. The DNA
double helix unwinds, and RNA synthesis
begins at the start point on the template strand.
Terminator:
Enhancer:
An RNA sequence that functions as a stop signal
-
Stimulates transcription
Location is not set
Affects rates of transcription (increase in rate)
Somehow works on RNA polymerase (protein-protein interactions)
Eukaryotic Cells Modify RNA After Transcription
-
Enzymes in the eukaryotic nucleus modify pre-mRNA in various ways before the
genetic messages are dispatched to the cytoplasm
-
Enzymes modify the two ends of a eukaryotic pre-mRNA molecule
A cap consisting of a modified GTP is added to the 5’ end of the RNA
A poly(A) tail consisting of up to 2000 adenine nucleotides is attached to the 3’
end, the end created by cleavage downstream of the AAUAAA termination signal
The modified ends help protect RNA from degradation
The poly(A) tail may facilitate the export of mRNA from the nucleus
When mRNA reaches the cytoplasm, the modified ends, in conjunction with certain
cytoplasmic proteins, signal a ribosome to attach to the mRNA
The leader and trailer segments of RNA are not translated
-
RNA Splicing
-
The removal of noncoding portions (introns) of the RNA molecule after initial
synthesis
Intron: A noncoding, intervening sequence within a eukaryotic gene (NOT IN
PROKARYOTES)
Exons: A coding region of a eukaryotic gene that is expressed. Exons are
separated from each other by introns
Ribozyme: An enzymatic RNA molecule that catalyzes reactions during RNA splicing
Spliceosome: A complex assembly that interacts with the ends of an RNA intron in
splicing RNA – releases an intron and joins two adjacent exons
Spliceosomes find binding sites by dinucleotide signals
EXON 1 – GU ~~~~~~~~~~~ Pyrimidine Tract – AG – EXON 2
-
The area between Exon 1 and Exon 2 is the intron segment
-
The area between Exon 1 and GU is the 5’ splice site, and the area between Exon 2
and AG is the 3’ splice site
Spliceosomes recognize GU, pyrimidine tract and AG, and that is how it knows what
needs to be spliced out
Thus, after splicing, the above example simply becomes EXON1 – EXON2
Alternative Splicing
-
purpose is to allow different proteins to be made from the same gene
gene expressed depends on how exons are ligated together (order must remain the
same – certain exons will be left out in alternative splicing)
found in G-protein coupled receptors – the final carboxy terminus can be
alternatively spliced
ie)
EXON 1 – EXON 2 – EXON 3 – EXON 4 – EXON 5
Alternative splicing examples:
EXON 1 – EXON 2 – EXON 4 – EXON 5
EXON 2 – EXON 5
EXON 3 – EXON 4 – EXON 5
* The order must remain the same –
example)
CAN NOT HAVE:
EXON 2 – EXON 1 – EXON 5 (for
EXON 5 – EXON1 – EXON 3 (for example)
This ends the section on transcription
Translation
Hampson did not talk too much about translation, but I’ll include the actual process –
he says we don’t really need to know it, but for what it’s worth, here’s translation.
-
In the process of translation, a cell interprets a genetic message and builds a
protein accordingly
Translation occurs outside the nucleus
Transfer RNA (tRNA)
tRNA: An RNA molecule that functions as an interpreter between nucleic acid and protein
language by picking up specific amino acids and recognizing the appropriate codons in
the mRNA
a) The 2-D structure of a general tRNA
molecule. There are four base pair
regions and 3 “loops”. At the 3’ end
of the molecule is the amino acid
attachment site, which has the same
base sequence for all tRNA’s. Within
the middle loop is the anticodon
triplet, which is unique to each tRNA
type. Note that although tRNA has
double stranded properties (4 sites of
base pair interactions), it is still a
single stranded RNA.
b) 3-D, L-shaped structure of tRNA (to
me, it looks more like an “r” than a
flipped “L” Note that the anticodon
loop and the amino acid attachment
site are quite distanced from each
other.
*Note: Anticodons are conventionally written 3’ to 5’ in order to align properly with
codons written 5’ to 3
-
A group of enzymes known as Aminoacyl – tRNA synthetase (there is at least one
for each amino acid) catalyze the attachment of an amino acid to its specific
tRNA molecule
Ribosomes
Ribosomal RNA (rRNA):
The most abundant type of RNA. Together with proteins, it
forms the structure of ribosomes that coordinate the sequential coupling of tRNA
molecules to the series of mRNA codons
Ribosoomes consist of a large subunit and a
small subunit. The large subunit has 3
sites for tRNA:
-
P site:
holds tRNA attached to growin polypeptide
-
A site:
holds tRNA carrying next amino acid to be added to chain
-
E site:
place where discharged tRNA leaves
Initiation of Translation
a) - small ribosomal subunit binds to a
molecule of mRNA
- An initiator tRNA, with the anticodon UAC,
base-pairs with the start
codon, AUG
- This tRNA carries the amino acid
methionine, which functions as the
“start” signal for the ribosome
*Hampson side note:
the start codon, not
- Although methionine is
all
codons begin with methionine
- This is due to proteolytic
processing –
proteins
are cleaved
- In prokaryotes, ShineDalgamo sequence
precedes
AUG start signal
- In eukaryotes, first
methionine at 5’ end is
start
signal (only one start site)
b) - The arrival of large ribosomal subunit
completes initiation complex
- Initiator tRNA is in the P site
- The A site is available to the tRNA bearing the next amino acid
Elongation
In elongation, amino acids are added one by one to the first amino acid.
step cycle:
Occurs in a 3
Termination
1) When the ribosome reaches the termination codon on a strand of mRNA, the A site
accepts a protein called a release factor – not tRNA
2) This release factor hydrolyzes the bond between the tRNA in the P site and last
amino acid of a polypeptide chain. The polypeptide is now free.
3) The two ribosomal subunits and the other components of the assembly dissociate
The Signal Mechanism for Targeting Proteins to the ER
- Signal peptide: A stretch of amino acids that target the polypeptide for the
endoplasmic reticulum
- The figure below shows the synthesis of a secretory protein and its simultaneous
import into the ER
1) Polypeptide synthesis begins on a free ribosome in the cytosol
2) A signal recognition particle (SRP) binds to the signal peptide
3) The SRP then binds to a receptor protein in the ER membrane. This receptor is
part of a protein complex (diagram one = translocation complex) that includes a
membrane pore and a signal cleaving enzyme
4) The SRP is released, and the growing polypeptide translocates across the
membrane. The signal peptide stays attached to the membrane
5) The signal-cleaving enzyme cuts off the peptide
6) The rest of the completed polypeptide leaves the ribosome and folds into its
final conformation
-
Hampson says that the signal peptide ensures that the polypeptide is oriented
properly
Review of Transcription, RNA modification, and Translation
And I think this covers all of the first three lectures. Again, it doesn’t cover
everything in the biochem textbook, but given we weren’t tested on the textbook for the
first test, I think this sufficiently covers everything we need to know. Again, I hope
this helps somewhat – only 2 more lectures, and no more biochem… until PHM 226 in
January =(