Transcript Molecular Genetics

Molecular Genetics
BACKGROUND:
• 1860 - Gregor Mendel determined
patterns of inheritance
• 1868 Friedrich Miescher discovered
material inside the cell nucleus
(chromosomes) is half protein and half
something else
• - other half later discovered to be DNA
(deoxyribonucleic acid)
• 1902 - Walter Sutton= genetic material
is found on chromosomes
Conclusion:
• 1. Chromosomes are made up of DNA
& Protein
• 2. Which one makes up the GENES?
Experiments to Determine
DNA or Protein
• 1. Frederick Griffith (1928): was
attempting to develop a vaccine against
pneumonia. He never succeeded but
did make some important discoveries
concerning DNA .
Griffith’s Experiment
 Took 2 strains of bacteria Streptococcus
pnuemoniae; inject them into mice in 4
different experiments:
• #1) Bacteria Enclosed in a smooth mucous
coat (smooth = S strain) = kill mice
• #2) Bacteria with Coat absent (rough = R
strain)= mice live
• #3) Heated strain S bacteria = made
harmless, mice lived
• #4) Mixed heated S strain with R strain =
MICE DIED!!
Conclusion:
• Transformation had taken place.
Transformation = process by which
bacterial cells incorporate DNA from
dead bacterial cells (transfer of genetic
information). The question remains: Is
DNA or protein portion of the
chromosome responsible for
transformation?
Avery, McCarty, & Macleod
• 2. Oswald Avery, Colin Macleod,
Maclyn McCarty (1944):
• Strong evidence for DNA as the
transforming principle.
• Used Enzymes: (repeated Griffith
experiment)
• Use a Protein destroying enzyme = transformation
still occurs
• Use a DNA
“
“ = NO transformation!
Hershey & Chase
• 3. Martha Chase & Alfred Hershey
(1952) : Proved DNA is the hereditary
material
• Used a Bacteriophage = a virus that
infects a bacteria cell; made of a DNA
core & protein coat; attached
radioactive labels (32P to DNA; 35S to
Protein) in two different batches.
 Viruses given time to attach to
bacteria and inject their genetic
material
 Separated the mixture using a high
speed centrifuge, this removes any
viral material remaining on the
outside
 35S radioactivity found only in liquid
 32P radioactivity found only in
bacteria
ALL NEW VIRUSES produced
in future generations
contained only radioactive 32P
• CONCLUSION: DNA and NOT protein
must be the genetic material!
The Structure of a DNA
Molecule
• Nucleotides = subunits of DNA; made
up of 3 components:
• 1. 5 - Carbon Sugar molecule
(deoxyribose)
• 2. Phosphate group
• 3. Nitrogen Base (4)
Nitrogenous Bases
• Purines
• - Adenine & Guanine
• - Double ring structure
• Pyrimidines
-
Cytosine & Thymine
- Single ring structure
Hydrogen bonds hold the
nitrogen bases together
Determining the Structure of a
DNA Molecule
• Erwin Chargaff (1950) : discovered in
cells that equal amounts of A & T and G
& C always exist.
• Chargaff’s Rule: A=T ; C=G (Purine
always bonded to a pyrimidine)
Determining the Structure of a
DNA Molecule
• Rosalind Franklin (1954) : used X-ray
diffraction to determine that DNA is a
long, thin molecule. She interpreted the
shape of a DNA molecule to be in the
shape of a helix (single coil)
Determining the Structure of a
DNA Molecule
• James Watson & Francis Crick (1962) :
determined the structure of a DNA
molecule to be in the shape of a Double
Helix (twisted ladder)
DNA STRUCTURE
 DNA molecule is made of COMPLEMENTARY
strands:
• one strand :
• Complement :
ATTG CAT
TAA C G TA
DNA STRUCTURE
 Twisted ladder structure:
• Sugar - Phosphate backbone = outside
rails of the ladder, held together by
strong covalent bonds
• Nitrogen Base Pairs = make up the
inside rungs (steps) held together by
weak hydrogen bonds
DNA Replication:
How does DNA make a copy of itself before Mitosis?
• Replication: process by which genetic
information gets copied such as during
Interphase of the cell cycle
 Involves separating “unzipping” the DNA
molecule into 2 strands
 Each strand serves as a template for making a
new complementary strand
 The process is SEMI CONSERVATIVE = each
new molecule consists of one new and one old
strand of DNA
 the sequence of bases gets preserved
Steps in the process of
Replication
• 1. Enzyme Helicase unwinds the DNA
helix (1A)
• 2. A Y-shaped Replication Fork results
(1B)
• 3. Single stranded DNA binding
proteins prevent the strands from
recombining (1C)
Steps in the Process of
Replication (cont.)
• 4. Topoisomerase removes any twists
or knots that form (1D)
• 5. RNA Primase initiates DNA
replication at special nucleotide
sequences called origins of
replication using RNA Primers
Steps in the Process of
Replication (cont.)
• 6. DNA Polymerase attaches to the
RNA primers and begins elongation =
adding DNA nucleotides to the
complement strand DNA polymerase
moves in the 3’  5’ direction along
each template (3)
Steps in the Process of
Replication (cont.)
• 7. The Leading Complementary
Strand ( 5’ 3’ ) is assembled
continuously (4)
• 8. The Lagging Complementary
Strand ( 3’ 5’ ) is assembled in short
Okazaki fragments which are joined by
DNA Ligase (5A, 5B)
Steps in the Process of
Replication (cont.)
• 9. RNA primers get replaced by DNA
nucleotides
Mutations: any sequence of nucleotides that
does not match the original DNA molecule from
which it was made
• Mutagen = anything that causes a
mutation to occur (UV light, radiation,
drugs, chemicals etc.)
 DNA can “proof read” itself
 DNA polymerase often does this
• Excision repair enzymes can fix mistakes
Types of Mutations
• Original DNA MESSAGE:
• THE DOG RAN AND THE FOX DID TOO
• Dna is read by the cell 3 base letters
(CODON) at a time, this is called a
Reading Frame
Point (substitution) = an
incorrect nucleotide
• THE HOG RAN AND THE FOX DID TOO
Deletion = missing nucleotide
• THE DOG RAN AND THE FOX DID TO
Insertion = additional
nucleotide is added
• THE DOG RAA NAN DTH EFO XDI
DTO O
• Frameshift mutation = reading frame is
every 3 bases (Codon)
Duplication = section of
nucleotides gets repeated
• THE DOG THE DOG THE DOG RAN
AND THE FOX DID TOO
Inversion = sequence of
nucleotides gets turned
around
• THE GOD RAN AND THE FOX DID TOO
Translocation = sequence of
nucleotides gets moved to
another chromosome
• THE DOG RAN AND THE CAT HAS
FUN ALL DAY
Protein Synthesis
 DNA in chromosomes contains genetic
instructions
 Those instructions regulate development,
growth, and metabolic activities.
 They also determine cell type and
characteristics
 DNA controls the cell by using codes of
Polypeptides (Proteins)
• Polypeptides (Proteins) = enzymes that
regulate chemical reactions or structural
components
• GENE (genotype) = genetic information
for a particular trait
• From a molecular viewpoint = traits are
the end product of metabolic processes
regulated by enzymes!
• The GENE is the DNA segment that
codes for a particular polypeptide
(protein) = One-gene-one-polypeptide
hypothesis
Protein Synthesis = process by which
enzymes and other proteins are manufactured
from the information contained in DNA
• Consists of three steps:
– 1. Transcription = transfer of information
from a strand of DNA to a strand of RNA
– 2. RNA Processing = modifies the RNA
molecule with deletions and additions
– 3. Translation = processed RNA used to
assemble amino acids into a polypeptide
3 types of RNA are involved in
the process:
• 1. Messenger RNA (mRNA) = carries
protein building instructions out of the
nucleus
• 2. Transfer RNA (tRNA) = carries amino
acids to ribosomes
• 3. Ribosomal RNA (rRNA) = building
blocks of ribosomes which coordinate
the activities of mRNA and tRNA
How is RNA Different from
DNA?
• RNA
 Is single stranded
 Bases = A, G, C and U (Uracil) replaces T
 Sugar = Ribose
CODON vs. ANTICODON
• Codon = a triplet group of 3 adjacent
nucleotides in mRNA; codes for one
specific amino acid
• Anticodon = a triplet group of 3
adjacent nucleotides in tRNA;
complementary to mRNA
PROTEIN SYNTHESIS
• Transcription:
• .Initiation = RNA polymerase attaches to
promoter regions on DNA and begins to unzip
the DNA into 2 strands. Promoter region
contains the sequence T-A-T-A (called the
TATA box)
• .Elongation = RNA nucleotides are assembled
using one side of the DNA molecule as a
template (5’ 3’)
• Termination = RNA polymerase reaches a
special sequence of nucleotides that serve as a
stop point; Usually AAAAAAA
PROTEIN SYNTHESIS
• Alterations take place before the mRNA
leaves the nucleus:
A 5’ Cap is added to the 5’ end of the molecule
5’ Cap = GTP (guanosine triphosphate)
This provides stability to the mRNA
Provides a point of attachment for the ribosome
(small unit)
Poly-A Tail added to the 3’ end
A sequence of 150 to 200 adenine nucleotides
The tail provides stability
Controls the movement of the mRNA across
the nuclear membrane
Some mRNA segments get removed
Exons = sequences that express a code for a
protein
Introns = intervening sequences that are
noncoding
SnRNPs (small nuclear ribonucleoproteins) =
delete out the introns and splice the exons
PROTEIN SYNTHESIS
• Translation:
• 1. Initiation = small ribosomal subunit attaches
to a special region near the 5’ end of the mRNA
• 2. A tRNA with the anticodon UAC attaches to
the mRNA start codon AUG
• 3. Large ribosomal subunit now attaches to the
mRNA
• 4. Elongation = tRNA’s deliver their amino
acids to the growing polypeptide
• 5. Ribosome moves over to the next codon and
repeats the process
• 6. Polypeptide chain elongates one amino acid
at a time
• 7. Termination = occurs when ribosome
encounters a stop codon
• The completed protein can now be
used by the cell as a structural unit
or as an enzyme!!
DNA Organization:
 DNA packaged with proteins forms a matrix
called Chromatin
 During cell division DNA = compact
Chromosomes
 Transposons = segments of DNA able to move
to new locations on the same chromosome or
to a different chromosome altogether
 Transposons have the effect of a mutation
Control of Gene Expression
 Every cell in a human contains the exact same
sequences of DNA
 Cells obviously have different functions
however
 Gene expression is regulated by the activation
then of only certain genes
• Example: gene regulation in E. coli
(well understood)
• OPERONS = sequence of DNA that
direct particular biosynthetic pathways.
4 Major Parts of an Operon
• 1. A regulatory gene produces a repressor
protein that prevents gene expression by
blocking the action of RNA polymerase
• 2. Promoter region of DNA attaches to RNA
polymerase to begin transcription
• 3. Operator region blocks the action of RNA
polymerase
• 4. Structural Genes contain DNA that codes
for several related enzymes that direct the
production of a product
Lac Operon = in E. coli
controls the breakdown of
Lactose
 Lactose is required to turn on the operon that
codes for the enzymes that break down
lactose.
 If lactose is not present the enzymes are not
made.