Tutorial - DNA

Watson & Crick

Griffith’s Experiments (Streptococcus pneumoniae)

Experiment Results Conclusion

1 R-Strain bacteria S-Strain bacteria

2 Heat-killed S-Strain

3 Heat-killed S-Strain+ R-Strain

Avery’s Experiments (enzymes & bacteria)

Nonvirulent strain of bacteria

Destroyed deadly bacteria

Some how the killed bacteria was able to pass “something” to the R-strain

That Transformed it to become deadly

Experiments Results Conclusion

Enzymes to break down proteins, carbohydrates, lipids, RNA, & finally DNA

DNA is the transforming molecule that made Griffith’s R-Strain bacteria turn

into an S-Strain bacteria

Hershey & Chase Experiments (bacteria & a virus called bacteriophage)Experiments Results Conclusion

Radioactive ProteinRadioactive DNA

Protein does not make new phagesDNA makes new phages

Mouse lived

Mouse lived

Mouse died

Deadly in spite of all enzymes except one that broke apart DNA

New phages = no radioactivity

Mouse died Virulent/deadly strain

New phages = radioactive

Anti-Parallel 5’ – 3’

DNA Structure:

1. Nucleosides

2. Nucleotides

3. Bases 1 hexagon ring+ 1 pentagon ring

1 hexagon ring

4. Chargaff’s Rule

PurinesAdenine, Guanine

PyrimidinesCytosine, Thymine

5’

5’3’

3’

A bonds to TC bonds to G

5. Franklin/Wilkins Watson/Crick

Conclusion-DNA is a helical structure With distinctive regularities.

OH

CH2

O

4

5

3 2

1

PO4

N base

deoxyribose

nucleotide

How would your replicatethis DNA molecule?

Which is the leading strand? Lagging strand? Which one has Okasaki Fragments?What enzymes are involved?What type of bonds are formed?What is the end result?

Replication fork

3’

5’

3’

5’

5’

3’

leading strand

Okazaki fragments

lagging strand

3’ 5’

DNA polymerase III

ligase

helicase

direction of replication

primase

DNA polymerase III

SSB

DNA polymerase I

Replication enzymes Helicase - unzips DNA

single-stranded binding proteins - controls the unzipping of DNA

DNA polymerase III - main DNA building enzyme

Primase - lays down RNA primer on lagging strand

DNA polymerase I - editing, repair & primer removal **

Ligase - “glues” Okazaki fragments together on lagging strand

Telomeres Expendable,

non-coding sequences at ends of DNA

short sequence of bases repeated 1000s times

TTAGGG in humans

Telomerase enzyme in certain cells

enzyme extends telomeres prevalent in cancers

Ends of chromosomes are eroded with each replication

an issue in aging? telomeres protect the ends of

chromosomes

Genetic Material

Prokaryotic DNA Eukaryotic DNA

• Circular in shape• In the cell’s cytoplasm• Not wrapped around proteins• Fewer average bp’s (base pairs)• No introns

• Linear in shape• In the cell’s nucleus• Wrapped around proteins• More bp’s (base pairs)• Introns and exons

What would happen if you put a eukaryotic DNA into a prokaryote?

DNA is tightly wound around histone proteins, making DNA inaccessible to enzymes that would code for the geneticinformation

Acetyl groups attach to the histonesCausing the tight compaction to unravel, now allowing DNA to be susceptible to activation (replication or transcription)

Regulating Gene Expression

A methyl group (CH3) can be attached to a cytosine base on DNA, as shown here. When a methyl group is attached to a base, the base cannot be accessed to build nucleotides

Implications: What effect would that have on the gene’s expression?

Ribosomes

Prokaryotic ribosomes Eukaryotic ribosomes

• 70S (smaller)• Synthesized and assembled in the cytoplasm• Simultaneous transcription and translation• Translation begins with f-met• Sensitive to antibiotics

• 80S (larger)• Synthesized in the nucleolus• Assembled in the cytoplasm (free) or (attached) on the    Rough Endoplasmic Reticulum• Transcription then translation• Translation begins with met

Both • Translation is powered by GTP (guanosine triphosphate) • Terminate translation with a stop codon & release factor proteins

proteinRNA

The “Central Dogma”

DNAtranscription translation

replication

flow of gene tic information within a cell

DNA - RNA - ProteinAll RNA’s (mRNA, rRNA, tRNA) are transcribed (made) in the nucleus

Transcription - the making of mRNA from a DNA template

RNA

Where is 5’ and 3’?

A A A A A3' poly-A tail

CH3

mRNA

5'

5' cap

3'

G PPP

Post-transcriptional processing Primary transcript

eukaryotic mRNA needs work after transcription

Protect mRNA from RNase enzymes in cytoplasm

add 5' cap add polyA tail

Edit out introns

eukaryoticDNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

primary mRNAtranscript

mature mRNAtranscript

pre-mRNA

spliced mRNA

50-250 A’s

Role of promoter1. Where to start reading

= starting point

2. Which strand to read

= template strand

3. Direction on DNA

= always reads DNA 3'5’

= transcribes DNA 5’3’

Transcription in Prokaryotes Initiation

RNA polymerase binds to promoter sequence on DNA

What do prokarotic mRNA lack in comparison to eukaryotic mRNA’s?

Ribosomes – made of rRNA and protein

P site (peptidyl-tRNA site) holds tRNA carrying growing

polypeptide chain

A site (aminoacyl-tRNA site) holds tRNA carrying next amino acid to

be added to chain

E site (exit site) empty tRNA

leaves ribosome from exit site

tRNA structure “Clover leaf” structure

anticodon on “clover leaf” end amino acid attached on 3' end

Building a polypeptide Initiation

brings together mRNA, ribosome subunits, proteins & initiator tRNA

Elongation Termination

Can you tell the story?

DNA

pre-mRNA

ribosome

tRNA

aminoacids

polypeptide

mature mRNA

5' cap

polyA tail

large subunit

small subunit

aminoacyl tRNAsynthetase

E P A

5'

3'

RNA polymerase

exonintron

tRNA

codon

Put it all together…

Lactose digestion in E.coli begins with its hydrolysis by the enzyme ß-galactosidase. The gene encoding ß-galactosidase, lacZ, is part of a coordinately regulated operon containing other genes required for lactose utilization.Which of the following figures correctly depicts the interactions at the lac operon when lactose is NOT being utilized? (The legend below defines the shapes of the molecules illustrated in the options.)

Lac OperonWhat’s it sound like it involves? Lac = Lactose; Operon = Operates when it’s On

Which of the following figures correctly depicts the interactions at the lac operon when lactose is NOT being utilized?

Implications to Genetically modified plants: a. Pest resistance? b. Herbicide resistance?