We have been studying patterns of inheritance for the last several weeks. We have used such terminology as traits and genes, but have not specifically targeted

the mechanism behind the transfer of those traits and genes from generation to

generation. Identify the material that provides the instructions for ALL of your traits and design an experiment to prove that this material is in fact the “stuff” of

what you are made.

Do Now

DNA! The stuff of Life

The beginning: Chromosomes related to phenotype

T.H. Morgan ◦ worked with Drosophila ◦ associated phenotype with

specific chromosome white-eyed male had specific X

chromosome

1908 | 1933

Morgan’s conclusions◦genes on chromosomes

◦BIG QUESTION – The Gene Wars: Is protein or DNA that are the genes?

Why all the fuss???

1928Frederick GriffithObserve the following data – explain

what is going on

Conclusion: heat-killed, virulent bacteria must have released genetic material transferred to R cells

Transformation – DNA from dead cells cut into fragments & exits cell → healthy cells

pick up free floating DNA and integrate chromosomes via

recombination

Oswald Avery

Maclyn McCarty

Colin MacLeod

Avery, McCarty & MacLeod - 1944

Avery, McCarty & MacLeod◦purified DNA & proteins separately from

Streptococcus pneumonia bacteria

Experimental Question: which will transform non-pathogenic bacteria?

◦1. injected protein into bacteria Mice lived!

◦2. injected DNA into bacteria transformed bacteria Mice died!

Hershey & Chase

Alfred HersheyMartha Chase

1952 | 1969

Protein coat labeledwith 35S DNA labeled with 32P

bacterial cells are agitatedto remove viral protein coats

35S radioactivityfound in the medium

32P radioactivity foundin the bacterial cells

Which radioactive marker is

found inside the cell?

This will be the molecule containing

genetic info!

bacteriophages infectbacterial cells

T2 bacteriophagesare labeled withradioactive isotopesS vs. P

Watson and Crick

1952

Rosalind Franklin & Maurice Wilkins

Chargaff DNA composition: “Chargaff’s rules”

◦varies from species to species◦all 4 bases not in equal quantity◦bases present in characteristic ratio humans:

A = 30.9% T = 29.4% G = 19.9% C = 19.8%

1947

RulesA = TC = G

Review with Hank!

The Cell Cycle

DNA Replication (Hank Video)

◦base pairing suggests that each side can serve as a template for a new strand

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” — Watson & Crick

* DNA Replication Machinery – 1:45

Prokaryotes: DNA usually circular(1 fork)

Eukaryotes: DNA linear(many forks)

A little more on DNA Replication…

Bacterial DNA is circular - Animation

Eukaryotic DNA is linear◦Can you think of any problems this may

pose in the successful completion of replication?

Animation

Telomeric Replication

A scientist is using an ampicillin-sensitive strain of bacteria that cannot use lactose because it has a nonfunctional gene in the lac operon. She has two plasmids. One contains a functional copy of the affected gene of the lac operon, and the other contains the gene for ampicillin resistance. Using restriction enzymes and DNA ligase, she forms a recombinant plasmid containing both genes. She then adds a high concentration of the plasmid to a tube of the bacteria in a medium for bacterial growth that contains glucose as the only energy source. This tube (+) and a control tube (-) with similar bacteria but no plasmid are both incubated under the appropriate conditions for growth and plasmid uptake. The scientist then spreads a sample of each bacterial culture (+ and -) on each of the three types of plates indicated below.

If no new mutations occur, it would be most reasonable to expect bacterial growth on which of the following plates?a. 1 and 2 onlyb. 3 and 4 onlyc. 5 and 6 onlyd. 4, 5, and 6 onlye. 1, 2, 3, and 4 only

If the scientist had forgotten to use DNA ligase during the preparation of the recombinant plasmid, bacterial growth would most likely have occurred on which of the following?

a. 1 and 2 only b. 1 and 4 onlyc. 4 and 5 onlyd. 1, 2, and 3 onlye. 4, 5, and 6 only

From Gene to Protein

sections of DNA that code for

proteins cells bodies

How does DNA code for cells & bodies?

DNA

The “Central Dogma” Flow of genetic information in a cell

◦ DNA to proteins?

transcriptiontranslation

replication

protein

RNA

DNA

trait

Metabolic Pathways… suggest genes code for enzymes

◦ Disruptions in pathways result in lack of an enzyme disease variation of phenotype

A B C D Edisease disease disease disease

metabolic pathway

Transcription in short…

Make a model!o Steps

o Structures

RNA review ribose sugar N-bases

◦ uracil instead of thymine◦ U : A◦ C : G

single stranded many RNAs

◦ mRNA, tRNA, rRNA, siRNA…

RNADNAtranscription

Making mRNA◦ transcribed DNA strand = template strand◦untranscribed DNA strand = coding strand◦synthesize complementary RNA strand

transcription bubble◦Enzymes involved

RNA polymerase Helicase

template strand

mRNA RNA polymerase

coding strand

DNA C C

C

C

C

C

C

C CC

G G G

G GG G

G

G

GAA

A A A

AA

AA

A A

A

ATT T

T

T

T

T

T

T T

T

T

U U

5

35

3

3

5build RNA 53

RNA polymerases 3 types of RNA polymerases

1. RNA polymerase 1 transcribe rRNA genes ONLY makes ribosomes

2. RNA polymerase 2 transcribe genes into mRNA

3. RNA polymerase 3 transcribe tRNA genes ONLY

**each has a specific promoter sequence it recognizes**

“What is a promoter?” you may ask Promoter region - site marking the start of gene ◦TATA box binding site

◦transcription factors (ie. proteins, hormones?) - on/off switch; trigger binding of RNA pol

◦RNA polymerase

Enhancer region◦binding site far upstream◦turns transcription on

HIGH

mRNA Processing Eukaryotic genes contain “fluff” – spliced

◦exons = expressed / coding DNA

◦introns = the junk; inbetween sequence; now thought to be involved in switches

5’ Cap & PolyA tail added

eukaryotic DNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

primary mRNA

transcriptmature mRNA

transcript

pre-mRNA

spliced mRNA

~10,000 bases

~1,000 bases

The splicing process…snRNPs

exonexon intronsnRNA

5' 3'

spliceosome

exonexcisedintron

5'

5'

3'

3'

3'

lariat

exonmature mRNA

5'

snRNPs “snurps”◦ small nuclear RNA◦ Proteins

Spliceosome◦ several snRNPs◦ recognize splice

site sequence cut & paste gene

The fancy cap & tail…

A A A A A3' poly-A tail

mRNA

5'5' cap

3'

G PPP

50-250 A’s

Enzymes in cytoplasm attack mRNA – protection is needed

add 5 GTP cap add poly-A tail

longer the tail, longer mRNA lasts: produces more protein

Translation

Make a model!o Steps

o Structures

How does mRNA code for proteins?TAC GCA CAT TTA CGT ACG CGG

DNA

AUG CGU GUA AAU GCA UGC GCC

mRNA

Met Arg Val Asn Ala Cys Ala

protein

?

How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?

4

4

20

ATCG

AUCG

20 different amino acids aa’s coded for by THREE

nucleotides –codons

4 bases, 3 per codon: 43 = 64 total possible combinations

Why don’t these numbers match? 20 amino acids, 64

options??WOBBLE

Code is redundant

◦several codons for each amino acid

◦3rd base “wobble”◦Most codons = aa’s

Start codon AUG methionine

Stop codons UGA, UAA, UAG

How are codons matched to amino acids?

A little more on tRNA “Clover leaf” structure

◦ anticodon on “clover leaf” end◦ amino acid attached to 3 end

Loading “naked” tRNA’s Aminoacyl tRNA synthetase - enzyme bonds

aa’s to tRNA◦ requires energy

ATP AMP bond is unstable can easily release amino acid at ribosome

activatingenzyme

anticodontRNATrp binds to UGG codon of mRNA

Trp Trp Trp

mRNA

C=OOH

OH H2OO

tRNATrp

tryptophan attached to tRNATrp

C=O

O

C=O

So where’s the protein making factory?

RIBOSOMES!!! Facilitate binding of

tRNA anticodon to mRNA codon

Organelle or enzyme?? Structure

◦rRNA & proteins◦2 subunits

large small

◦3 sites

3 ribosomal sites… A site (aminoacyl-tRNA site)

◦ tRNA carrying next aa to be added to chain binds here

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

polypeptide chain

E site (exit site)◦ empty tRNA

leaves ribosome from exit site

Met

UUA C

A G

APE

Met

3'

UUA C

A G

APE

5'

Let’s translate… Initiation

◦brings together mRNA, ribosomal subunits, initiator tRNA

Elongation◦adding amino acids

based on codon sequence

Termination◦end codon

123

Full circle: metabolic pathways

Do you think this process is the same for prokaryotes & eukaryotes? Explain

your ideas.

Prokaryotes◦DNA in

cytoplasm◦circular

chromosome◦naked DNA◦no introns◦continuous

process

Eukaryotes◦DNA in nucleus◦linear

chromosomes◦DNA wound on

histone proteins◦introns vs. exons◦mRNA

processing

Translation in Prokaryotes Transcription & translation simultaneous in

bacteria ◦ DNA in cytoplasm◦ no mRNA editing ◦ ribosomes read

mRNA as transcribed

◦ Faster than in eukaryotes (DNA to protein ~1hr)

Transcription mRNA processing

mRNA splicing

Review Videos

Translation Animation Protein Synthesis

Let’s review