Download DNA and PROTEIN SYNTHESIS

DNA and PROTEIN SYNTHESIS
“Cracking the Code”
DNA
• the blueprint of life
• contains the instructions for making
proteins within the cell.
• Deoxyribonucleic Acid
• Found in the nucleus of the cell
(mitochondria also have DNA)
The Shape of DNA
• very long polymer
• a ‘twisted ladder’ or zipper
• called a double helix.
Prokaryotic vs Eukaryotic DNA
• Prokaryotic- found free moving in the
cytoplasm, not contained by a nucleus.
DNA tends to be circular in plasmid.
• Eukaryotic – found contained inside of the
nucleus, DNA tends to be linear, and uses
histones to coil it tightly.
Structure of DNA
• A 5 carbon SUGAR
(deoxyribose)& phosphate
backbone
• rungs that connect the
backbones are nitrogenous
bases
• Nucleotide = sugar + phosphate
+ nitrogenous base
– This structure was discovered by
Structure of DNA
Anti parallel: one strand runs 5’ to
3’, the other runs opposite
4 DNA Bases
•
Purine base - double
rings.
– Adenine
– Guanine
•
Pyrimidine base single ring
– Thymine
– Cytosine
Complementary Pairing
• complementary base pairs: normal pairing
of nitrogenous bases is: 1 purine and 1
pyrimidine
• Adenine always and only binds with Thymine
A-T
• Guanine always and only pairs with Cytosine
G-C
-These ratios was discovered by Erwin
Chargoff
DNA Replication
• semi-conservative replication-new DNA
molecule made of one parent and one newly
replicated strand. (Meselson and Stahl)
• In general a DNA molecule ‘unzips’ down the
middle of the paired bases, 2 individual strands
are made that will become the ‘templates’ for
new complete DNA stands
The Steps for DNA Replication (during Sphase of Interphase):
1. Initiation starts at a specific necletide
sequence, a group of enzymes called DNA
helicases breaks hydrogen bonds between
bases to unzip the double helix
2. Proteins bind to keep strands apart
3) RNA primers attach to a spot on the
original DNA stand
4) DNA polymerase III – starts at where the
primer attached to the DNA and makes
new strand in 5’ to 3’ direction (always)
-DNA polymerase III can only be added to a 3’ end
5) DNA polymerase 1 – removes primers
and replaces with nucleotide
6) DNA ligase – joins DNA fragments
DNA makes DNA
DNA Editing
• AMAZING!!! DNA has a spell check
• DNA polymerase cut out mismatched
base, and replace it with the proper
nucleotides.
Protein Synthesis
• Proteins -chains of amino acids.
• as small as 8 amino acids, and as large as over
50,000 amino acids.
• There are 20 amino acids
• One Gene One Protein Theory - production of
each protein is controlled by one gene. (The
sequence of nucleotides in a gene will determine
the structure/type/role of the protein) This is the
CENTRAL DOGMA of DNA that Watson
described
(Protein Function)
Functions:
- Pigments – eg.
Melanin – protect
or signal etc.
(Protein Function)
- enzymes – eg.
Amylase – catalyze
reactions
(Protein Function)
- movement – eg.
Actin in muscle
(Protein Function)
• carriers – eg.
Hemoglobin
(oxygen)
(Protein Function)
channels – in
membranes (eg.
Porin)
(Protein Function)
•
- receptors –
cell recognition eg.
CD4 receptor on
WBC (AIDS)
Essential amino acids
Ribonucleic acid (RNA)
-required for protein synthesis,
1. RNA is single stranded, DNA is double stranded
2. The 5 carbon sugar is ribose in RNA,
deoxyribose in DNA
3. RNA uses the base uracil instead of thymine.
Uracil binds with adenine. (A-U )
3 Types of RNA
1. Messenger RNA (mRNA)
•
takes DNA code to the ribosomes where proteins
are made
2. Transfer RNA (tRNA)
•
brings the appropriate amino acids from the
cytoplasm to the ribosomes and strings them in
order according to mRNA.
3. Ribosomal RNA (rRNA)
• not directly involved with protein synthesis
• makes the ribosomes in the nucleus
Protein Synthesis
Steps in Protein Synthesis
1. Transcription
• At a start sequence of neucleotides, the
double stranded DNA opens up
(unzipped by helicases again)
• RNA polymerase attaches to the
promoter and builds the single stranded
mRNA
• Stops at the termination sequence of
nucleotides and mRNA detaches
• mRNA makes its way to the ribosome for
translation
2. Translation-making the protein
• mRNA attaches between the two
subunits of a ribosome which “reads”
the mRNA
–
mRNA is a codon (represents a 3 nucleotide
sequence from the DNA that it just read)
• tRNA brings amino acids from the
cytoplasm to the ribosome
– tRNA has an anticodon for an amino acid
ex) If mRNA codes UUU, t RNA anticodon is
AAA and it will get the amino acid PHE
mRNA Codons
• 3 nitrogen bases that code for a specific
amino acid –WHY 3?
• Start codon – starts protein synthesis,
AUG which codes for methionine
• Stop codons – ends protein synthesis,
UAA, UAG, UGA
• Intron – non-coding sequences
• Other non-coding regions of DNA are: telomeres,
regulators, stop codon.
• Exon – coding sequences that make
proteins
• Once translation is complete tRNA returns
to the cytoplasm and mRNA is broken
down.
Changes or additions to
transcription
• Methylation: The adding of a -CH3 (methyl)
group to a cytosine on some area of the DNA
strand. This methyl group now prevents RNA
polymerase from transcribing any region past
the methyl blockage. Usually involved in cell
specialization
• Poly (aaa) tail- At the end of transcription a long
section adenines can be added to the 3’ end
(‘Tail’) of the mRNA strand that adds stability to
the mRNA strand. These adenines prevent
degradation of the mRNA stand
Why Do Firefly’s Glow?
• What Makes a Firefly Glow?
DNA and Mutations
• inheritable changes
• Spontaneous or caused by
mutagenic agents
– Ex) radiation, chemicals
• may be as simple as a single base
pair
Basic Mutations:
1. Substitution of nucleotide(s), usually mild
mutation created (but can be serious like sickle
cell anemia)
2. Frameshift – changes the reading frame. A whole
new sequence is read, usually leads to severe
mutations. Frameshifts are caused by:
a) Deletion of a nucleotide(s)
b) Addition of extra nucleotide(s)
1. Translocation of a gene-DNA fragment switches
location, often between different chromosomes.
This is a very serious mutations (usually fatal)
• Silent mutations: A mutation in the intron regions
(non-coding sequences of DNA) or a mutation
that causes a nitrogen base to change but not
cause any change in amino acid produced.
Usually due to a substitution mutation
• Missense mutation: a mutation that changes the
DNA sequence so that a different amino acid is
coded. Usually due to a substitution mutation
• A Nonsense mutation: a mutation that results in
a stop codon being produced which results in no
amino acids after the stop codon from being
produced. Usually lethal
• Mutations in body cells often have little
consequences compared to mutations of
the germ cells (sperm or egg).
• A single mistake in the DNA of a sperm or
egg cell would be repeated billions of
times if that cell underwent fertilization to
become a complete individual.
Transposons
• are “jumping genes”,
DNA sequences that
have the ability to
move in / out of
chromosomes,
changing their
location. May cause
mutations or change
amount of DNA
• http://www.dnalc.org/vshockw
ave/ac_ds_trans.dcr
Oncogenes and Cancer
Cancer -uncontrolled cell division from a changed
genetic code (uncontrolled mitosis)
Evidence:
1. Cancerous cells often display nitrogen
base substitutions
2. Many known mutagens, are also known to
cause cancer
3. segments of chromosomes extracted
from cancerous mice transformed normal
mouse cells into cancerous cells.
-genes called oncogenes cause cancer
Oncogenes and Cancer
• oncogenes are present in normal cells, and do
not always result in cancer
• oncogenes must be transposed to another site
on the chromosome to be activated
The Ames Test
• To test for potential mutagens
• bacteria Salmonella typhimurium - unable to
make histidine
• after the bacteria is exposed to a potential
mutagen, it is grown in a culture without
histidine.
• If it survives it has been mutated!
Biotechnology
• Biotechnology –biological systems used to
produce a product.
• Genetic engineering –produces transgenic
cells: foreign DNA inserted
• Recombinant DNA – DNA that comes
from 2 or more sources.
• Vector –an organism (bacteria or virus) that
carries and leaves its genetic material in a host
cell. The host then replicates the vector’s genetic
material.
• Plasmid – is circular DNA found in bacteria. This
is often used as a vector in DNA recombination.
The process of creating recombinant
DNA involves 2 types of enzymes:
1. Restriction Enzymes (aka restriction
endonucleases) – (DNA scissors) cut the
DNA strand at specific sites -palindromes- and
often creates sticky ends
2. The now unpaired segments can be
paired with complimentary nucleotides
from a different strand of DNA
3. DNA ligase – is genetic glue that puts
DNA strands back together.
•
rDNA
Application
• Insulin used to be harvested from pigs, but some
individuals did not tolerate it.
• we are now able to use bacteria to produce
human insulin in large quantities.
• is tolerated much better by patients.
• This procedure was first marketed in Canada in
1983!
Other Aspects of Biotechnology
• Gene sequencing –determining the
specific location and composition of
specific genes.
• The Human Genome Project -1990
2000: mapped our 30 000 genes (3
billion nucleotides)
• NOVA Online | Cracking the Code of Life |
Sequence for Yourself
• RFLP – restriction fragment length
polymorphism –cut DNA into pieces
• Gel Electrophoresis – separate the DNA
pieces by size.
• DNA fingerprinting
– identify an individual based on their
unique genetic code.
– homologous segments of DNA are
similar but contain unique patterns of
nitrogen bases
– identify individuals involved in crimes,
paternity
DNA Fingerprinting
Misc. Technology
• Polymerase chain reaction –making
billions of copies of a piece of DNA- PCR
Animation
– We now use Taq from Thermus aquaticus bacterium to do PCR
as it is resistanct to denaturing during the application of heat
during PCR
• Paternity Testing - Paternity Testing
• Gene Gun - Genegun1