Title: Announcements
1Announcements
- Pick up lab overview for transformation lab this
week. - Homework-problem set 4- due in lab this week.
- 3. Look over Ch. 11, problems 4, 5, and 8 for
exam 2. - 4. Group B presentations are coming up 10/29, 30
start thinking of topics and deciding on sources.
Group A did very well - pressure is on! - 5. Review session in class Wednesday. Bring your
questions! - 6. Exam 2 next week 10/17, 18, and 20. 15
multiple choice and 7 written (exam 1 had 18
multiple choice and 9 written). Exam is at CLAS
testing center, available 3 days this time. Hours
are 9-9 Thursdays, 9-5 Fridays, and 3-7 Sundays.
Bring a pencil, bluebook, calculator. - 7. Summer scholar program - research opportunity
in summer, 2400 stipend. Need to find a faculty
member to sponsor you. Often need to volunteer in
the lab spring semester. Application includes a
formal written proposal deadline mid-Feb. need
56 credits completed by start of summer and be
returning next fall to CMU.
2Review of Last Lecture
- Evidence that DNA is genetic material
- Structure of DNA/RNA 5 different bases, 2
different sugars, phosphates - History the race to determine the structure of
DNA was VERY competitive 2 key pieces of data
Chargaffs base compostion analysis and X-ray
diffraction studies
3Outline of Lecture 20
- I. Structure of DNA
- II. Analytical analysis of nucleic acids
- III. Replication of DNA
How is DNA organized? 1 single chain, 2 chains,
3 chains? How does the structure allow for
replication, expression, storage and mutation?
4I. The DNA Double Helix
- DNA structure
- Double helical
- major, minor grooves
- right-handed
- bases are 3.4 Å apart (10 Å 1 nm)
- 10 bases/turn
- Complementary Base Pairing
- through H bonds AT, G?C
- Antiparallel Strands
- 5 to 3
- 3 to 5
Discussion of original paper in class Friday
5Right- and Left-handed DNA
6Base-Pairing in DNA
AT
G?C
7Structure of RNA
Sugar ribose, not 2-deoxyribose Bases
uracil, not thymine Organization
single-stranded, not double-stranded
How is genetic information in DNA expressed?
First step is transcribing RNA from DNA -
single-stranded RNA is generated using DNA as a
template
8Reading DNA Strands
Single strand of DNA 5-AGCATTCG-3 3-TCGTAAGC-
5 Complementary strand of above, usually written
5 to 3 5-CGAATGCT-3 Double-stranded
fragment is written 5-AGCATTCG-3 3-TCGTAAGC-5
9Learning Check
The sequence of the dwarf gene in garden peas is
as follows
5 - A G C T A C G T -3 3 - T C G A T G C A -5
Write the RNA sequence transcribed from the top
strand of DNA, 5- 3.
10II. Analytical analyses of nucleic acids
Denaturation/Renaturation
Determining the Tm allows for an estimate of the
base composition of a DNA sample
1
2
Which DNA has higher GC content and why?
11Nucleic Acid Hybridization
Transcription of 1 strand of DNA 3
G G T T G G G C C A A C C C
A C G C T T G C G A
2
1
U U U G C G C
T T T G C G C A AA C G C G
3
Add RNA to denatured DNA allow to hybridize
Heat - denature
A C G C T T G C G A
G G T T G G G
G G T T G G G C C A A C C C
A C G C T
2
1
C C A A C C C
T G C G A
U U U G C G C
Hybrid
T T T G C G C
A A A C G C G
3
A AA C G C G
12Nucleic Acid Gel Electrophoresis
13What makes nucleic acids acidic?
Base Pairing Rules
14Points to know about DNA structure
- Note how many hydrogen bonds are in the base
pairing - If 2, then the pair is AT
- If 3, then the pair is GC
- Recall that A and G are purines with 2 rings,
while T and C are pyrimidines with 1 ring also T
has a CH3 group on its ring.
15III. DNA Replication
How is genetic information replicated accurately
at each cell division? Could each strand of the
DNA double helix act as a template for the
complementary strand?
At each cell division, 109 base pairs are
replicated. If error rate is 10-6 , then 3000
errors/cell division - TOO many.
16DNA Replication is Semiconservative
17Other Theoretical Possibilities
18Separation of Nucleic Acids by CeCl Gradient
Centrifugation
19Meselson-Stahl Experiment
DNA Labeling with 15N
Subsequent Generations Labeled with 14N
Cesium Chloride Gradient Banding
20Expected Results From Conservative or Dispersive
Reproduction
If Conservative Two bands, heavy and light, in
1st and 2nd generations
If Dispersive, one smeary band in 1st and
2nd generations
21Expected Results if Semiconservative
These results were obtained. A related
experiment was performed in plants (Fig. 12.5)
22Bacterial DNA Replication begins at a Single
Origin and Proceeds Bidirectionally
Origin of Replication
23DNA Polymerase I can Synthesize DNA
- Arthur Kornberg et al. (1957) discovered the
enzyme in E. coli - Requires template DNA strand, primer, MgCl2, and
4 dNTPs - Monomers added 5 to 3
245 to 3 Addition of Monomers
25DNA polymerases I, II and III
- pol I
- most abundant (400/cell)
- RNA primer removal
- pol II
- unknown abundance
- DNA repair?
- pol III
- low abundance (15/cell)
- DNA replication
26Problems of DNA Synthesis
- Unwinding
- Tension must be relieved
- Priming
- Antiparallel strands
- RNA primer removal
- Backbone joining
- Proofreading
27Steps of DNA Synthesis
- Denaturation and Unwinding
- Priming and Initiation
- Continuous and Discontinuous Synthesis
- Including Proofreading and Error Correction
- Removal of Primer
- Ligation of nicks in backbone
28Steps of DNA SynthesisDenaturation and
Unwinding of DNA
- DnaA, DnaB, DnaC proteins are helicases which
bind origin and separate strands - Single-strand binding protein (SSBP) keeps
strands apart - DNA gyrase, a type of DNA topoisomerase, cuts to
relax supercoiling
29Initiation of Synthesis
- RNA Primase makes RNA primer on DNA template
- DNA Polymerase III extends primer with DNA
- DNA Polymerase I removes RNA primer, replaces
with DNA
30Directionality of DNA synthesis
31Proofreading occurs as polymerase moves along
if incorrect base pairing, base is removed and
replaced.
32Continuous and Discontinuous Synthesis
- Continuous
- on Leading Strand.
-
- Discontinuous
- on Lagging Strand
- creates Okazaki
- fragments.
- DNA ligase joins
- nicks in backbone.