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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
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???
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
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!
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
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
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?
transcription
translation
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 E
disease disease disease diseasemetabolic pathway
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
DNAC
C
C
C
C
C
C
C CC
GG
G
G G
G G
G
G
GAA
AA A
A
A
A
A
A A
A
AT
T 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 intron
snRNA
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 AA
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
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
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=O
OHOH
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
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
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