announcements 1. pick up lab overview for transformation lab this week. 2. homework-problem set 4-...

Announcements1. Pick up lab overview for transformation lab this week.

2. 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.

Review of Last Lecture

1. Evidence that DNA is genetic material

2. Structure of DNA/RNA: 5 different bases, 2 different sugars, phosphates

3. History: the race to determine the structure of DNA was VERY competitive; 2 key pieces of data = Chargaff’s base compostion analysis and X-ray diffraction studies

Outline 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?

I. 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: A=T, GC

• Antiparallel Strands– 5’ to 3’– 3’ to 5’

Discussion of original paper in class Friday

Right- and Left-handed DNA

Base-Pairing in DNA

A=T

GC

Structure 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

Reading 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’

Learning 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’.

Denaturation/Renaturation

Which DNA has higher GC content and why?

II. Analytical analyses of nucleic acids

1 2

Determining the Tm allows for an estimate of the base composition of a DNA sample

A C G C T T G C G A

G G T T G G GC C A A C C C

T T T G C G CA AA C G C G

1 2

3

U U U G C G C

Transcription of 1 strandof DNA 3

A C G C T

T G C G A

G G T T G G G

C C A A C C C

T T T G C G C

A AA C G C G3

21

Heat - denature

U U U G C G CA A A C G C G

Add RNA to denatured DNA;allow to hybridize

Hybrid

A C G C T T G C G A

G G T T G G GC C A A C C C

Nucleic Acid Hybridization

Nucleic Acid Gel Electrophoresis

Base Pairing RulesWhat makesnucleic acidsacidic?

Points 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.

III. 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.

DNA Replication is Semiconservative

Other Theoretical Possibilities

Separation of Nucleic Acids by CeCl Gradient Centrifugation

Meselson-Stahl Experiment

DNA Labeling with 15N

Subsequent Generations Labeled with 14N

Cesium Chloride Gradient Banding

Expected Results From Conservative or Dispersive Reproduction

If Conservative: Twobands, heavy and light,in 1st and 2nd generations

If Dispersive, one smearyband in 1st and 2ndgenerations

Expected Results if Semiconservative

These results were obtained.

A related experiment was performed in plants (Fig. 12.5)

Bacterial DNA Replication begins at a Single Origin and Proceeds Bidirectionally

Origin ofReplication

DNA 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’

5’ to 3’ Addition of Monomers

DNA 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

Problems of DNA Synthesis

• Unwinding

• Tension must be relieved

• Priming

• Antiparallel strands

• RNA primer removal

• Backbone joining

• Proofreading

Steps 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

Steps of DNA Synthesis:Denaturation 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

Initiation 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

Directionality of DNA synthesis

Proofreadingoccurs as polymerasemoves along; ifincorrect base pairing, base is removed and replaced.

Continuous and Discontinuous Synthesis

• Continuouson Leading Strand. • Discontinuouson Lagging Strandcreates Okazakifragments.

• DNA ligase joinsnicks in backbone.