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AP Biology
AP Biology 2017
How Genes
Work
Organelles
nucleus
ribosomes
endoplasmic reticulum (ER)
Golgi apparatus
vesicles
Making proteins
smallribosomalsubunit
largeribosomalsubunit
cytoplasm
mRNA
nuclear pore
Nucleus & NucleolusNucleolus
Function
ribosome production
build ribosome subunits from rRNA & proteins
exit through nuclear pores to cytoplasm &
combine to form functional ribosomes
smallsubunit
large subunit
ribosome
rRNA &proteins
nucleolus
smallsubunit
largesubunitRibosomes
Function
protein production
Structure
rRNA & protein
2 subunits combine 0.08mm
Ribosomes
RoughER
SmoothER
membrane proteins
Types of Ribosomes
Free ribosomes
suspended in cytosol
synthesize proteins that
function in cytosol
Bound ribosomes
attached to endoplasmic
reticulum
synthesize proteins
for export or
for membranes
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DNA
RNA
ribosomes
endoplasmic
reticulum
vesicle
Golgi
apparatus
vesicle
proteinon its way!
protein finishedprotein
Making Proteins
TO:
TO:
TO:
TO:
nucleus
TO:
End of the Tour
TACGCACATTTACGTACGCGGATGCCGCGA
CTATGATCACATAGACATGCTGTCAGCTCT
AGTAGACTAGCTGACTCGACTAGCATGATC
GATCAGCTACATGCTAGCACACYCGTACAT
CGATCCTGACATCGACCTGCTCGTACATGC
TACTAGCTACTGACTCATGATCCAGATCAC
TGAAACCCTAGATCGGGTACCTATTACAGT
ACGATCATCCGATCAGATCATGCTAGTACA
TCGATCGATACTGCTACTGATCTAGCTCAA
TCAAACTCTTTTTGCATCATGATACTAGAC
TAGCTGACTGATCATGACTCTGATCCCGTA
What happens in the cellwhen a gene is read?
Where are the genes?
Where does a gene start?Where does the gene end?
How do cells make proteinsfrom DNA?
How is one gene read and another one not?
How do proteinscreate phenotype?
Inheritance of metabolic diseases
suggested that genes coded for enzymes
each disease (phenotype) is caused by
non-functional gene product
lack of an enzyme
Tay sachs
PKU (phenylketonuria)
albinism
Am I just the sum of my proteins?
Metabolism taught us about genes
A B C D E
disease disease disease disease
enzyme 1 enzyme 2 enzyme 3 enzyme 4
metabolic pathway
ingested protein
phenylalanine
tyrosine
hydroxyphenylpyruvicacid
homogentisicacid
maleylacetoaceticacid
CO2 & H2O
phenylalanine hydroxylase
transaminase
hydroxyphenylpyruvic acidoxidase
homogentisic acidoxidase
melanin
thyroxine
PKUphenylketonuria
tyrosinosis
alkaptonuria
albinism
cretinism
digestion
1 gene – 1 enzyme hypothesis
Beadle & Tatum
Compared mutants of bread mold,
Neurospora fungus
created mutations by X-ray treatments
X-rays break DNA
damage a gene
wild type grows on minimal media
sugars + required nutrients allows fungus to
synthesize essential amino acids
mutants require added amino acids
each type of mutant lacks a certain enzyme needed
to produce a certain amino acid
non-functional enzyme from damaged gene
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Wild-typeNeurospora
Minimalmedium
Select one ofthe spores
Grow oncomplete medium
Minimalcontrol
Nucleicacid
CholinePyridoxine RiboflavinArginine
Minimal media supplemented only with…
ThiamineFolicacid
NiacinInositolp-Aminobenzoic acid
Test on minimalmedium to confirmpresence of mutation
Growth oncompletemedium
X rays or ultraviolet light
asexualspores
spores
Beadle & Tatum
create mutations
positive control
negative control
experimentals
amino acidsupplements
One gene / one enzyme hypothesis
chromosome
genecluster 1
enzyme E
glutamate ornithine citruline argino-succinate
arginine
enzyme F enzyme G enzyme H
encoded
enzyme
substrate inbiochemical pathway
genecluster 2
genecluster 3
arg-Harg-Garg-Farg-E
Damage to specific gene, mapped to
nutritional mutations
gene thatwas damaged
Beadle & Tatum 1941 | 1958
George Beadle
Edward Tatum
"for their discovery that genes act by
regulating definite chemical events"
one gene : one enzyme hypothesis
The “Central Dogma”
Flow of genetic information in a cell
How do we move information from DNA to proteins?
replication
proteinRNADNA trait
DNA gets all the glory,
but proteins do all the work!
RNA
ribose sugar
N-bases
uracil instead of thymine
U : A
C : G
single stranded
lots of RNAs
mRNA, tRNA, rRNA, siRNA…
RNADNAtranscription
AP Biology 2017
Transcription
from
DNA nucleic acid language
to
RNA nucleic acid language
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Transcription
Making mRNA transcribed DNA strand = template strand
untranscribed DNA strand = coding strand same sequence as RNA
synthesis of complementary RNA strand transcription bubble
enzyme RNA polymerase
template strand
rewinding
mRNARNA polymerase
unwinding
coding strand
DNAC C
C
C
C
C
C
C
C CC
G
GG
G
G G
G G
G
G
GA
A
AA A
A
A
A
A
A A
A
AT
T T
T
T
T
T
T
T T
T
T
U U
5
3
5
3
3
5build RNA 53 AP Biology 2017
Transcription
in Prokaryotes
Bacterial chromosome
mRNA
Cell wall
Cellmembrane
Transcription
Psssst…no nucleus!
Transcription in Prokaryotes
Initiation
RNA polymerase binds to promoter
sequence on DNA
Role of promoter
Starting point
where to start reading
start of gene
Template strand
which strand to read
Direction on DNA
always read DNA 35
build RNA 53
Transcription in Prokaryotes
Promoter sequences
RNA polymerase
molecules bound to
bacterial DNA
TATAAT
RNA polymerase
Promoter
enzymesubunit
bacterial DNA
–35 sequence –10 sequenceTTGACA
RNA polymerasestrong vs. weak promoters
read DNA 35
Transcription in Prokaryotes
Simple proofreading
1 error/105 bases
make many mRNAs
mRNA has short life
not worth editing!
Elongation
RNA polymerase
copies DNA as it
unwinds
~20 base pairs at a time
300-500 bases in gene
builds RNA 53
reads DNA 35
Transcription in Prokaryotes
Termination
RNA polymerase stops at termination sequence
RNA GC
hairpin turn
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AP Biology 2017
Transcription in Eukaryotes
Protein
RNA Processing
Translation
Transcription
Psssst…DNA can’t
leave nucleus!
Prokaryote vs. Eukaryote genes
Prokaryotes
DNA in cytoplasm
circular
chromosome
naked DNA
no introns
Eukaryotes
DNA in nucleus
linear
chromosomes
DNA wound on
histone proteins
introns vs. exons
eukaryotic
DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
intronscome out!
Transcription in Eukaryotes
3 RNA polymerase enzymes
RNA polymerase 1
only transcribes rRNA genes
makes ribosomes
RNA polymerase 2
transcribes genes into mRNA
RNA polymerase 3
only transcribes tRNA genes
each has a specific promoter sequence
it recognizes
Transcription in Eukaryotes
Initiation complex
transcription factors bind
to promoter region
upstream of gene
suite of proteins which bind
to DNA
turn on or off transcription
TATA box binding site
recognition site for
transcription factors
transcription factors
trigger the binding of RNA
polymerase to DNA
AmRNA
5'
3'
GPPP
Post-transcriptional processing
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
primary mRNAtranscript
mature mRNAtranscript
pre-mRNA
spliced mRNA
Primary transcript (pre-mRNA)
eukaryotic mRNA needs work after transcription
mRNA processing (making mature mRNA)
mRNA splicing = edit out introns
protect mRNA from enzymes in cytoplasm
add 5 cap
add polyA tail
~10,000 bases
~1,000 bases
1977 | 1993
Richard Roberts Philip
SharpCSHL
MITadenovirus
common cold
Discovery of Split genes
beta-thalassemia
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Splicing must be accurate
No room for mistakes!
splicing must be exactly accurate
a single base added or lost throws off the
reading frame
AUG|CGG|UCC|GAU|AAG|GGC|CAU
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
Met|Arg|Ser|Asp|Lys|Gly|His
Met|Arg|Val|Arg|STOP|
Splicing enzymes
snRNPs
exonexon intron
snRNA
5' 3'
spliceosome
exonexcisedintron
5'
5'
3'
3'
3'
lariat
exonmature mRNA
5'
No, not smurfs!“snurps”
snRNPs
small nuclear RNA
proteins
Spliceosome
several snRNPs
recognize splice
site sequence
cut & paste
Whoa! I think we just broke
a biological “rule”!
Ribozyme
Sidney Altman Thomas Cech
1982 | 1989
Yale U of Colorado
RNA as ribozyme
some mRNA can even splice itself
RNA as enzyme
AP Biology
Translation
from
nucleic acid language
to
amino acid language
AP Biology
Translation in
Prokaryotes
Bacterial chromosome
mRNA
Cell wall
Cellmembrane
Transcription
Translation
proteinPsssst…no nucleus!
Transcription & translation are simultaneous
in bacteria
DNA is in
cytoplasm
no mRNA
editing
ribosomes
read mRNA
as it is being
transcribed
Translation in Prokaryotes
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Translation: prokaryotes vs. eukaryotes
Differences between prokaryotes & eukaryotes
time & physical separation between processes takes eukaryote ~1 hour
from DNA to protein
RNA processing
AP Biology
Translation in Eukaryotes
mRNA
From gene to protein
DNAtranscription
nucleus cytoplasm
mRNA leaves nucleus through nuclear pores
proteins synthesized by ribosomes using instructions on mRNA
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
ribosome
proteintranslation
How does mRNA code for proteins?
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
Met Arg Val Asn Ala Cys Alaprotein
?
How can you code for 20 amino acids
with only 4 nucleotide bases (A,U,G,C)?
4
4
20
ATCG
AUCG
AUGCGUGUAAAUGCAUGCGCCmRNA
mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
Met Arg Val Asn Ala Cys Alaprotein
?
codon
Cracking the code1960 | 1968
Crick
determined 3-letter (triplet) codon system
Nirenberg & Khorana
WHYDIDTHEREDBATEATTHEFATRATWHYDIDTHEREDBATEATTHEFATRAT
Nirenberg (47) & Khorana (17)
determined mRNA–amino acid match
added fabricated mRNA to test tube of
ribosomes, tRNA & amino acids
created artificial UUUUU… mRNA
found that UUU coded for phenylalanine (phe)
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AP Biology
1960 | 1968Marshall Nirenberg
Har Khorana
The code Code for ALL life!
strongest support for
a common origin for
all life
Code is redundant
several codons for
each amino acid
3rd base “wobble”
Start codon
AUG
methionine
Stop codons
UGA, UAA, UAG
Why is thewobble good?
How are the codons matched to
amino acids?
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
aminoacid
tRNA
anti-codon
codon
5 3
3 5
3 5
UAC
MetGCA
ArgCAU
Val
mRNA
From gene to protein
DNAtranscription
nucleuscytoplasm
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
ribosome
proteintranslation
aa
Transfer RNA structure
“Clover leaf” structure
anticodon on “clover leaf” end
amino acid attached on 3 end
Loading tRNA Aminoacyl tRNA synthetase
enzyme which bonds amino acid to tRNA
bond requires energy
ATP AMP
energy stored in tRNA-amino acid bond unstable
so it can release amino acid at ribosome easily
activatingenzyme
anticodon
tRNATrp binds to UGG condon of mRNA
Trp Trp Trp
mRNAA C CUGG
C=O
OHOH
H2OO
tRNATrp
tryptophan attached to tRNATrp
C=O
O
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Ribosomes
Facilitate coupling of
tRNA anticodon to
mRNA codon
organelle or enzyme?
Structure
ribosomal RNA (rRNA) & proteins
2 subunits
large
smallE P A
Ribosomes
Met
5'
3'
UUA C
A G
APE
A site (aminoacyl-tRNA site)
holds tRNA carrying next amino acid to
be added to chain
P site (peptidyl-tRNA site)
holds tRNA carrying growing
polypeptide chain
E site (exit site)
empty tRNA
leaves ribosome
from exit site
Building a polypeptide
Initiation brings together mRNA, ribosome
subunits, initiator tRNA
Elongation adding amino acids based on
codon sequence
Termination end codon 123
Leu
Leu Leu Leu
tRNA
Met MetMet Met
PE A
mRNA5' 5' 5' 5'
3' 3' 3'3'
U UA AAAC
CC
AU UG GGU
UA
AAAC
CC
AU UG GG
UU
AAA
ACC
C
AU UG GGU U
AAAC
CA U UG G
ValSer
AlaTrp
releasefactor
AA A
CCU UGG 3'
Protein targeting
Signal peptide
address label
Destinations: secretion
nucleus
mitochondria
chloroplasts
cell membrane
cytoplasm
etc…start of a secretory pathway
Can you tell
the story?
DNA
pre-mRNA
ribosome
tRNA
aminoacids
polypeptide
mature mRNA
5' cap
polyA tail
large ribosomal subunit
small ribosomal subunit
aminoacyl tRNAsynthetase
E P A
5'
3'
RNA polymerase
exon intron
tRNA
AP Biology 2007-2008
Got Questions?
Can I translate that for you?
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COLD STORAGE
Old Slides
Translation
Codons
blocks of 3
nucleotides
decoded into
the sequence
of amino acids
From nucleus to cytoplasm…
Where are the genes?
genes are on chromosomes in nucleus
Where are proteins synthesized?
proteins made in cytoplasm by ribosomes
How does the information get from DNA
in nucleus to cytoplasm?
messenger RNA
nucleus
Alternative splicing Alternative mRNAs produced from same gene
when is an intron not an intron…
different segments treated as exons
Starting to gethard to
define a gene!
Domains
Modular architecture
of many proteins
exons may represent
functional units of
protein
easier to mix and match
in the production of new
proteins?
So… What is a gene? One gene – one enzyme?
but not all proteins are enzymes
but all proteins are coded by genes
One gene – one protein?
but many proteins are composed of several polypeptides
but each polypeptide has its own gene
One gene – one polypeptide?
but many genes only code for RNA (tRNA, rRNA…)
One gene – one product?
but many genes code for
more than one product …
So…Where doesthat leave
us?!
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Defining a gene…
“Defining a gene is problematic because… one gene can code for several protein products, some genes code only for RNA, two genes can overlap, and there are many other complications.”
– Elizabeth Pennisi, Science 2003
gene
polypeptide 1
polypeptide 2
polypeptide 3
RNAgeneIt’s hard to
hunt for wabbits,if you don’t knowwhat a wabbit
looks like.
TACGCACATTTACGTACGCGGATGCCGCGACTATGATC
ACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACT
CGACTAGCATGATCGATCAGCTACATGCTAGCACACYC
GTACATCGATCCTGACATCGACCTGCTCGTACATGCTA
CTAGCTACTGACTCATGATCCAGATCACTGAAACCCTA
GATCGGGTACCTATTACAGTACGATCATCCGATCAGAT
CATGCTAGTACATCGATCGATACTGCTACTGATCTAGC
TCAATCAAACTCTTTTTGCATCATGATACTAGACTAGC
TGACTGATCATGACTCTGATCCCGTAGATCGGGTACCT
ATTACAGTACGATCATCCGATCAGATCATGCTAGTACA
TCGATCGATACTGCTACTGATCTAGCTCAATCAAACTC
TTTTTGCATCATGATACTAGACTAGCTGACTGATCATG
ACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGA
TCATCCGATCAGATCATGCTAGTACATCGATCGATACT
human genome
3.2 billion bases
AAAAAAAAGTP
20-30b
3'
promoter transcription
stop
transcription
start
introns
The Transcriptional unit (gene?)
transcriptional unitTAC ACT
DNA
DNATATA5'RNA
polymerase
pre-mRNA
5' 3'
translation
start
translation
stop
mature mRNA
5' 3'
UTR UTR
exonsenhancer
1000+b20-30b
3'
introns
The Transcriptional unit
transcriptional unitTAC ACT
DNATATA5'RNA
polymerase
5' 3'
5' 3'
exonsenhancer
1000+b