Division Ave. High School
Ms. Foglia
AP Biology
Making proteins
From Gene
to Protein
Organelles
nucleus
ribosomes
endoplasmic reticulum
(ER)
Golgi apparatus
vesicles
How Genes
Work
nuclear pore
small
ribosomal
subunit
mRNA
large
ribosomal
subunit
AP Biology
2007-2008
AP Biology
Nucleus & Nucleolus
cytoplasm
Nucleolus
Function
ribosome production
build ribosome subunits from rRNA & proteins
exit through nuclear pores to cytoplasm &
combine to form functional ribosomes
large subunit
rRNA &
proteins
small
subunit
AP Biology
large
subunit
Ribosomes
Function
small
subunit
protein production
Free ribosomes
rRNA & protein
2 subunits combine
0.08µ
µm
suspended in cytosol
synthesize proteins that
function in cytosol
Bound ribosomes
Ribosomes
nucleolus
Types of Ribosomes
Structure
ribosome
AP Biology
Rough
ER
attached to endoplasmic
reticulum
synthesize proteins
for export or
for membranes
Smooth
ER
AP Biology
AP Biology
membrane proteins
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Division Ave. High School
Ms. Foglia
AP Biology
TO:
endoplasmic
reticulum
nucleus
protein
on its way!
DNA
RNA
TO:
End of the Tour
vesicle
TO:
TO:
vesicle
TO:
ribosomes
finished
protein
protein
Golgi
apparatus
Making Proteins
AP Biology
AP Biology
TAC G C A C ATTTAC GTAC G C G G AT G C
C G C G A CTATG ATCA C ATA G A C AT G C
What happens in the cell
TGTCA G CTCTA GTA
GaAgene
CTA
G CT G A C
when
is read?
TC G A CTA G C AT G ATC G ATCA G CTA C
Where are the genes?
AT
G CTA G C A C A CYC GTA C ATC G ATC
Where does a gene start?
end?GTAC AT G C
CT G A C ATC GWhere
A Cdoes
CTtheG gene
CTC
How do cells make proteins
from DNA?
TACTA G CTA CT G A CTCAT G ATCC
AG
ATCA CT G A A A C C CTA
G ATC
How is one
gene readG G GTA C
and another one not?
CTATTAC A GTA C G ATCATCC G ATCA
How
do proteins
G ATC AT G CTA GTAC ATC G
ATC
G ATA
create phenotype?
AP Biology
CT G CTA CT G ATCTA G CTC A ATCA A A
1941 | 1958
Beadle & Tatum
one gene : one enzyme hypothesis
Metabolism taught us about genes
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
metabolic pathway
A
Am I just the
sum of my proteins?
disease
disease
disease
disease
B
C
D
E
AP Biology enzyme 1
enzyme 2
enzyme 3
enzyme 4
One gene / one enzyme hypothesis
Damage to specific gene, mapped to
nutritional mutations
gene
cluster 1
gene
cluster 2
gene
cluster 3
chromosome
arg-E
encoded
enzyme
George Beadle
Edward Tatum
AP Biology
"for their discovery that genes act by
regulating definite chemical events"
enzyme E
glutamate
arg-G arg-H
arg-F
enzyme F
ornithine
substrate in
APbiochemical
Biology
pathway
enzyme G
citruline
enzyme H
arginosuccinate
arginine
gene that
was damaged
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Division Ave. High School
Ms. Foglia
AP Biology
The “Central Dogma”
RNA
Flow of genetic information in a cell
How do we move information from DNA to proteins?
DNA
cripti
trans
on
RNA
uracil instead of thymine
U:A
C : G
n
latio
trans
protein
trait
DNA gets
all the glory,
but proteins do
all the work!
replication
ribose sugar
N-bases
single stranded
lots of RNAs
mRNA, tRNA, rRNA, siRNA…
transcription
DNA
AP Biology
RNA
AP Biology
Transcription
Making mRNA
transcribed DNA strand = template strand
untranscribed DNA strand = coding strand
synthesis of complementary RNA strand
enzyme
Transcription
from
DNA nucleic acid language
to
RNA nucleic acid language
same sequence as RNA
transcription bubble
5′′
A
G
T
A T C
T A
C
DNA
G
3′′
AP Biology
2007-2008
AP Biology
build
RNA 5′′→ 3′′
Bacterial chromosome
A G C
G
A T
C G T
A
C
T
3′′
G C A U C G U
C
G T A G C A
T
T
A
A
C
T
A G
C T
G
A
T
unwinding
3′′
5′′
rewinding
mRNA
5′′
RNA polymerase
template strand
Transcription in Prokaryotes
Transcription
in Prokaryotes
coding strand
RNA polymerase
Initiation
Transcription
RNA polymerase binds to promoter
sequence on DNA
mRNA
Role of promoter
Psssst…
no nucleus!
Cell
membrane
Direction on DNA
always read DNA 3′′ →5′′
APBiology
build RNA 5′′→ 3′′
Cell wall
AP Biology
Starting point
where to start reading
start of gene
Template strand
which strand to read
2007-2008
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Division Ave. High School
Ms. Foglia
AP Biology
Transcription in Prokaryotes
Transcription in Prokaryotes
Promoter sequences
Elongation
enzyme
subunit
RNA polymerase
read DNA 3′′→5′′
bacterial DNA
RNA polymerase
copies DNA as it
unwinds
Promoter
TTGACA TATAAT
–35 sequence
–10 sequence
~20 base pairs at a time
300-500 bases in gene
builds RNA 5′′→ 3′′
Simple proofreading
RNA polymerase
molecules bound to
bacterial DNA
RNA polymerase
strong vs. weak promoters
AP Biology
1 error/105 bases
make many mRNAs
mRNA has short life
not worth editing!
AP Biology
reads DNA 3′′ → 5′′
Transcription in Prokaryotes
Transcription in Eukaryotes
Termination
RNA polymerase stops at termination
sequence
Transcription
RNA GC
hairpin turn
Psssst…
DNA can’t
leave nucleus!
RNA Processing
Translation
Protein
AP Biology
AP Biology
Prokaryote vs. Eukaryote genes
Prokaryotes
Eukaryotes
DNA in cytoplasm
circular
chromosome
naked DNA
no introns
Transcription in Eukaryotes
DNA in nucleus
linear
chromosomes
DNA wound on
histone proteins
introns vs. exons
introns
come out!
3 RNA polymerase enzymes
only transcribes rRNA genes
makes ribosomes
RNA polymerase 2
RNA polymerase 3
each has a specific promoter sequence
it recognizes
exon = coding (expressed) sequence
AP Biology
RNA polymerase 1
intron = noncoding (inbetween) sequence
eukaryotic
DNA
2007-2008
transcribes genes into mRNA
only transcribes tRNA genes
AP Biology
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Division Ave. High School
Ms. Foglia
AP Biology
Transcription in Eukaryotes
Post-transcriptional processing
Initiation complex
mRNA processing (making mature mRNA)
suite of proteins which bind
to DNA
turn on or off transcription
TATA box binding site
recognition site for
eukaryotic mRNA needs work after transcription
mRNA splicing = edit out introns
protect mRNA from enzymes in cytoplasm oly-A tail 3'
3' p
AA
add 5′′ cap
A A A
’s
mRNA
50 A
cap
50-2
'
5
P
add polyA tail
5' G PP
intron = noncoding (inbetween) sequence
~10,000 bases
transcription factors
Primary transcript (pre-mRNA)
transcription factors bind
to promoter region
upstream of gene
eukaryotic DNA
transcription factors
trigger the binding of RNA
polymerase to DNA
exon = coding (expressed) sequence
pre-mRNA
primary mRNA
transcript
AP Biology
AP Biology
~1,000 bases
mature mRNA
transcript
spliced mRNA
Bacterial chromosome
Translation in
Prokaryotes
Translation
from
nucleic acid language
to
amino acid language
Transcription
mRNA
Translation
Psssst…
no nucleus!
protein
Cell
membrane
Cell wall
AP Biology
2007-2008
AP Biology
2007-2008
Translation in Prokaryotes
Translation: prokaryotes vs. eukaryotes
Transcription & translation are simultaneous
in bacteria
DNA is in
cytoplasm
no mRNA
editing
ribosomes
read mRNA
as it is being
transcribed
AP Biology
Differences between prokaryotes &
eukaryotes
time & physical separation between
processes
takes eukaryote ~1 hour
from DNA to protein
RNA processing
AP Biology
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Division Ave. High School
Ms. Foglia
AP Biology
From gene to protein
Translation in Eukaryotes
aa
aa
aa
transcription
DNA
2007-2008
How does mRNA code for proteins?
DNA
AP Biology
aa
aa
mRNA
mRNA leaves
nucleus through
nuclear pores
AP Biology
translation
protein
aa
aa
aa
ribosome
proteins synthesized
by ribosomes using
instructions on mRNA
nucleus
cytoplasm
mRNA codes for proteins in triplets
TAC G C A C ATTTAC GTAC G C G G
4 ATCG
DNA
mRNAA U G C G U G U A A A U G C A U G C G C C
4 AUCG
TAC G C A C ATTTAC GTAC G C G G
codon
?
protein
Met Arg ValAsn Ala Cys Ala
mRNAA U G C G U G U A A A U G C A U G C G C C
?
20
How can you code for 20 amino acids
with only 4 nucleotide bases (A,U,G,C)?
AP Biology
Cracking the code
1960 | 1968
protein
AP Biology
Marshall Nirenberg
1960 | 1968
Nirenberg & Khorana
Crick
determined 3-letter (triplet) codon system
W H Y DIDTHERED B ATEATTHEFATRAT
Met Arg ValAsn Ala Cys Ala
Har Khorana
Nirenberg (47) & Khorana (17)
determined mRNA–amino acid match
added fabricated mRNA to test tube of
ribosomes, tRNA & amino acids
AP Biology
created artificial UUUUU… mRNA
found that UUU coded for phenylalanine (phe)
AP Biology
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Division Ave. High School
Ms. Foglia
AP Biology
The code
strongest support for
a common origin for
all life
How are the codons matched to
amino acids?
Code for ALL life!
DNA
Code is redundant
several codons for
each amino acid
3rd base “wobble”
5′′
3′′
codon
3′′
Start codon
AP Biology
5′′
mRNA
AUGC GU GUAAAU GCAU GC GCC
Why is the
wobble good?
3′′
TAC G C A C ATTTAC GTAC G C G G
AUG
methionine
5′′
tRNA
UAC
amino
acid
Met
From gene to protein
Transfer RNA structure
aa
“Clover leaf” structure
aa
aa
transcription
DNA
Val
AP Biology
UGA, UAA, UAG
C A U anti-codon
Arg
Stop codons
GCA
aa
translation
anticodon on “clover leaf” end
amino acid attached on 3′′ end
aa
mRNA
protein
aa
aa
aa
ribosome
aa
AP Biology
nucleus
cytoplasm
Loading tRNA
Ribosomes
Aminoacyl tRNA synthetase
enzyme which bonds amino acid to tRNA
bond requires energy
energy stored in tRNA-amino acid bond
AP Biology
Facilitate coupling of
tRNA anticodon to
mRNA codon
Structure
ATP → AMP
unstable
so it can release amino acid at ribosome easily
organelle or enzyme?
ribosomal RNA (rRNA) & proteins
2 subunits
Trp C=O
O
Trp
H2O
O
activating
enzyme
=O
C
OH
OH
Trp C=O
tRNATrp
anticodon
AP Biology
tryptophan attached
to tRNATrp
AC C
U GG
large
small
E P A
mRNA
tRNATrp binds to UGG
condon of mRNA
AP Biology
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Division Ave. High School
Ms. Foglia
AP Biology
Ribosomes
A site (aminoacyl-tRNA site)
empty tRNA
leaves ribosome
from exit site
Elongation
adding amino acids based on
codon sequence
holds tRNA carrying growing
polypeptide chain
Termination
Met
3 2 1
end codon
E site (exit site)
brings together mRNA, ribosome
subunits, initiator tRNA
holds tRNA carrying next amino acid to
be added to chain
P site (peptidyl-tRNA site)
Initiation
Leu
Val
Met
Met
Met
Met Leu
release
factor
Ser
Ala
Leu
Leu
Trp
tRNA
C
U A C
A U G
5'
E
P
A
Building a polypeptide
G
A
AP Biology
3'
U A C U G AA U
5'
C
mRNA A U G
3'
E P A
5'
U A C G A C AA
AU G C U G U
U A C GA C
AU G C U G AAU
5'
3'
3'
5'
U A C G A C AA U
AU G C U G
3'
A CC
U GG U A A
3'
AP Biology
RNA polymerase
Destinations:
Protein targeting
Signal peptide
address label
start of a secretory pathway
secretion
nucleus
mitochondria
chloroplasts
cell membrane
cytoplasm
etc…
DNA
Can you tell
the story?
amino
acids
exon
intron
tRNA
pre-mRNA
5' cap
mature mRNA
aminoacyl tRNA
synthetase
polyA tail
large ribosomal subunit
polypeptide
5'
small ribosomal subunit
AP Biology
AP Biology
3'
tRNA
E P A
ribosome
Got Questions?
Can I translate that for you?
Substitute Slides
for Student Print version
AP Biology
2007-2008
AP Biology
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2007-2008
8
Division Ave. High School
Ms. Foglia
AP Biology
Can you tell
the story?
Extra Slides
(used some years & not others)
AP Biology
AP Biology
Translation
Building Proteins
Codons
2007-2008
Organelles involved
nucleus
ribosomes
endoplasmic reticulum
(ER)
Golgi apparatus
vesicles
blocks of 3
nucleotides
decoded into
the sequence
of amino acids
The Protein Assembly Line
nucleus
AP Biology
Where are the genes?
genes are on chromosomes in nucleus
Where are proteins synthesized?
How does the information get from DNA
in nucleus to cytoplasm?
AP Biology
ER
Golgi
apparatus
vesicles
AP Biology
From nucleus to cytoplasm…
ribosome
Alternative splicing
Alternative mRNAs produced from same gene
when is an intron not an intron…
different segments treated as exons
proteins made in cytoplasm by ribosomes
Starting to get
hard to
define a gene!
messenger RNA
nucleus
AP Biology
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Division Ave. High School
Ms. Foglia
AP Biology
Domains
So… What is a gene?
Modular architecture
of many proteins
One gene – one enzyme?
exons may represent
functional units of
protein
easier to mix and match
in the production of new
proteins?
One gene – one protein?
One gene – one polypeptide?
One gene – one product?
AP Biology
Defining a gene…
“Defining a gene is problematic because…
because…
one gene can code for several protein products,
some genes code only for RNA, two genes can
complications.””
overlap, and there are many other complications.
– Elizabeth Pennisi, Science 2003
gene
RNA
polypeptide 1
gene
polypeptide 2
AP Biology
polypeptide 3
It’s hard to
hunt for wabbits,
if you don’t know
what a wabbit
looks like.
but many proteins are composed of several polypeptides
but each polypeptide has its own gene
AP Biology
but not all proteins are enzymes
but all proteins are coded by genes
but many genes only code for RNA (tRNA, rRNA…)
but many genes code for
more than one product …
So…
Where does
that leave
us?!
TAC G C A CATTTAC GTAC G C G G AT G C C G C G A CT
AT G ATCAC ATA G A CAT GCT GTCA G CTCTA GTA
genome
G A CTA G CT human
G ACTC G A CTA
G C AT G ATC G ATCA G
CTACAT G CTA
G
C
A
CA
CYC
GTAC
ATC G ATCCT G
3.2 billion bases
AC ATC G A C CT G CTC GTACATG CTACTA G CTAC
TGA CTCAT GATCC A G ATCA CT G AA AC C CTA G A
TC G G GTAC CTATTACA GTAC G ATCATCC G ATC
A G ATCAT G CTA GTACATC G ATC G ATACT GCTA
CT G ATCTA G CTCA ATCAA ACTCTTTTTGCATCA
TGATACTA G A CTA G CT G ACT GATCAT G A CTCT
G ATCCC GTA G ATC G G GTACCTATTACA GTAC G
ATCATCC G ATCA G ATCAT G CTA GTACATC G AT
C G ATACT G CTACT G ATCTA G CTCA ATCAA ACT
CTTTTTGCATCAT
G ATACTA G A CTA G CT GA CT
AP Biology
G ATCAT GA CTCTG ATCCC GTA G ATC G G GTAC C
The Transcriptional unit (gene?)
enhancer
1000+b
20-30b
3'
RNA
TATA
polymerase
translation
start
TAC
Any Questions??
translation
stop
exons
transcriptional unit
What color would a smurf turn
if he held his breath?
5'
DNA
ACT
DNA
UTR
promoter
UTR
introns
transcription
start
transcription
stop
5'
pre-mRNA
AP Biology
5'
GTP mature mRNA
3'
3'
AAAAAAAA
AP Biology
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10
Division Ave. High School
Ms. Foglia
AP Biology
The Transcriptional unit
enhancer
exons
1000+b
20-30b
3'
TAC
RNA
TATA
polymerase
transcriptional unit
5'
DNA
ACT
introns
Richard
Roberts
Philip
Sharp
MIT
CSHL
5'
5'
3'
beta-thalassemia
AP Biology
Splicing must be accurate
No room for mistakes!
splicing must be exactly accurate
a single base added or lost throws off the
reading frame
A U G C G G CTATG G G U C C G A U A A G G G C C A U
AUGCGGUCCGAUAAG GGCCAU
A U G|CG G|UCC|GAU|AA G|GGC|CAU
Met|Arg|Ser|Asp|Lys|Gly|His
A U G C G G CTATG G G U C C G A U A A G G G C C A U
AUGCGG GUCCGAUAAGG GCCAU
A U G|CG G|GUC|CGA|UAA|GG G|CCA|U
AP Biology
Met|Arg|Val|Arg|STOP|
Ribozyme
Whoa! I think
we just broke
a biological “rule”!
Splicing enzymes
snRNPs
adenovirus
common cold
3'
AP Biology
1977 | 1993
Discovery of Split genes
small nuclear RNA
exon
proteins
Spliceosome
exon
3'
spliceosome
5'
3'
cut & paste
No,
not smurfs!
“snurps”
AP Biology
intron
5'
several snRNPs
recognize splice
site sequence
snRNPs
snRNA
mature mRNA
lariat
5'
exon
5'
3'
exon
3'
excised
intron
1982 | 1989
RNA as ribozyme
some mRNA can even splice itself
RNA as enzyme
Sidney Altman
AP Biology
Yale
Thomas Cech
U of Colorado
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