Molecular genetics
• Previous discussions focused on the individual.
• Focus has now shifted to genes
•
•
•
•
•
How are they encoded (3/29, 4/2)
How are they replicated (4/2, 4/5)
How are they expressed - transcription (4/7)
How are they expressed - translation (4/9)
Relationship between phenotype and genotype - pathways
(4/12)
• How are they regulated (4/14, 4/16, 4/19)
• How we study them - individual genes (4/19, 4/21, 4/23)
• Review 4/28 and Exam 4/30
• How we study them - global studies (Genomics-4/26, 5/3,
5/5)
Important Dates in Determining the Structure of DNA
1
Why did they do
the experiment?
2
How did they do
the experiment?
3
What did they
find out?
4
What did they
learn?
Model question
• One interpretation of the results obtained by
Griffith after performing experiment (d, previous
slide) is stated as the “conclusion” in previous
slide.
State one other possible interpretation? What
experiments and conclusion did they use to rule
out this interpretation?
Review the findings that were used by
Watson and Crick to propose the double
helix model for the structure of DNA
– Erwin Chargaff’s data
– Rosiland Franklin’s x-ray crystallography
data
Structure of DNA
• DNA is composed of four basic
molecules; nucleotides
– Nucleotides contain:
• Phosphate
• Sugar, deoxyribose
– Ribose in RNA
• One of four nitrogenous bases (two purines
and two pyrimidines)
5’-end
3’-end
3’-end
(free 3’-OH)
5’-end
(free 5’phosphate)
The two polynucleotide
strands (the
backbones) in the
double helix run in
opposite directions,
and are said to be antiparallel
Because of the pairing
(A-T; G-C), one
polynucleotide chain is
always complementary
to the base sequence of
the other strand
Model questions
The strand of a DNA mo lecule has the base
sequence 3Υ-GCCTTTAAG-5Υ. Upon replication
the complementary base sequence on the other
strand of D NA will be _____________.
Which of the following is NOT a key feature of DNA
molecule
a. antiparallel
b. minor groove
c. major groove
d. double helix
*e. stems and loops
Matthew Meselson and Franklin Stahl, 1958
entirely new
AND entirely
old DNA
molecules
present
ALL DNA
molecules
are made up
of both old
and new DNA
entirely new
DNA molecules
present BUT
not entirely old
DNA molecules
What Meselson and Stahl expected if
semiconservative replication
First generation
results helped
them rule out
one of the three
possible mode
of replication
Matthew Meselson and Franklin Stahl, 1958
entirely new
AND entirely
old DNA
molecules
present
ALL DNA
molecules
are made up
of both old
and new DNA
entirely new
DNA molecules
present BUT
not entirely old
DNA molecules
Clincher evidence!
Why?
In dispersive model
lighter DNA band
should not have
formed
Great test question:
Predict what the
cesium chloride
gradients would look
like for conservative
and dispersive
replication!
2
3
1
1 = initiator proteins
2 = single strand binding proteins
3 = helicase
4 = topoisomerase (gyrase)
4
Model questions
The existe nce of le ading and lagging strands during DNA re plication is the result of the
___________________.
a. need for an RNA primer to start DNA synthesis
b. formation of Okazaki fragments
*c. fact that DNA poly merase can synthesize only in the 5Υto 3Υdirection
d. semi conservative nature of DNA replication
e. None of these.
Replication
• DNA Polymerase III
– Synthesize new DNA in the 5’  3’ direction
• Synthesizes long sequences of new DNA
• Is highly processive; synthesizes DNA for a long period of time
without releasing the template
• For example, synthesizes leading strand
• DNA Polymerase I
– Synthesize new DNA in the 5’  3’ direction
• Only synthesizes short sequences of new DNA
• But before it could do this, it needs to remove RNA primers
• This is achieved by its 5’  3’ exonuclease activity
Replication
• DNA Polymerase I
– Synthesize new DNA in the 5’  3’ direction
• Only synthesizes short sequences of new DNA
– 3’  5’ exonuclease activity (proofreading)
– 5’  3’ exonuclease activity (remove primers)
• DNA Polymerase III
– Synthesize new DNA in the 5’  3’ direction
• Synthesizes long sequences of new DNA
– 3’  5’ exonuclease activity (proofreading)
NOTE: DNA polymerase III does not have the
5’  3’ exonuclease activity
5’ 3’
exonuclease
activity of
DNA
polymerase I
Model question
You know that DNA ligase seals the gaps left behind by DNA
polymerase I and completes DNA replication. You also
know that both enzymes form the phosphodiester bonds. If that
is the case why do cells need DNA ligase to seal the nicks and
why not they use DNA polymerase I to seal these nicks?