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Division Ave. High School
Ms. Foglia
AP Biology
From Gene
to Protein
How Genes
Work
AP Biology
2007-2008
What do genes code for?
n
How does DNA code for cells & bodies?
u
how are cells and bodies made from the
instructions in DNA
DNA
proteins
cells
bodies
AP Biology
The “Central Dogma”
n
Flow of genetic information in a cell
u
How do we move information from DNA to proteins?
DNA
replication
RNA
protein
trait
DNA gets
all the glory,
but proteins do
all the work!
AP Biology
1
Division Ave. High School
Ms. Foglia
AP Biology
Metabolism taught us about genes
n
Inheritance of metabolic diseases
u suggested that genes coded for enzymes
u each disease (phenotype) is caused by
non-functional gene product
Am I just the
sum of my proteins?
lack of an enzyme
Tay sachs
n PKU (phenylketonuria)
n albinism
n
n
metabolic pathway
û
A
disease
disease
disease
disease
B
C
D
E
AP Biology enzyme 1
û
enzyme 2
û
enzyme 3
û
enzyme 4
1941 | 1958
Beadle & Tatum
one gene : one enzyme hypothesis
George Beadle
Edward Tatum
"for their discovery that genes act by
regulating definite chemical events"
AP Biology
Beadle & Tatum
X rays or ultraviolet light
Wild-type
Neurospora
create mutations
asexual
spores
Minimal
medium
spores
Growth on
complete
medium
positive control
Select one of
the spores
Test on minimal
medium to confirm
presence of mutation
negative control
Grow on
complete medium
Minimal media supplemented only with…
experimentals
Choline
Pyridoxine
Riboflavin
Minimal
Nucleic
Arginine
control
Niacin
amino acid p-Amino
Inositol acid Folic
supplements
acid
benzoic acid
Thiamine
AP Biology
2
Division Ave. High School
Ms. Foglia
AP Biology
a
a
From gene to protein
nucleus
DNA
cytoplasm
transcription
mRNA
a
a
translation
a
a
a
a
a
a
a
a
a
a
a
protein
a
a
a
a
a
a
a
ribosome
trait
AP Biology
Transcription
from
DNA nucleic acid language
to
RNA nucleic acid language
AP Biology
2007-2008
RNA
n
n
ribose sugar
N-bases
uracil instead of thymine
U:A
uC : G
u
u
n
n
single stranded
lots of RNAs
u
DNA
AP Biology
mRNA, tRNA, rRNA, siRNA…
transcription
RNA
3
Division Ave. High School
Ms. Foglia
AP Biology
Transcription
n
Making mRNA
u
transcribed DNA strand = template strand
untranscribed DNA strand = coding strand
u
synthesis of complementary RNA strand
u
enzyme
u
n
n
n
same sequence as RNA
transcription bubble
coding strand
RNA polymerase
5¢
C
DNA
G
3¢
AP Biology ¢® ¢
build
RNA 5 3
A
G
T
A T C
T A
G
C
A
G C
T
C
G
T
5¢
A
3¢
G C A U C G U
C
G T A G C A
rewinding
mRNA
A
T
T
A
T
RNA polymerase
A
C
T
A G
C T
G
A
T
unwinding
3¢
5¢
template strand
RNA polymerases
n
3 RNA polymerase enzymes
u
RNA polymerase 1
n
n
only transcribes rRNA genes
makes ribosomes
u
RNA polymerase 2
u
RNA polymerase 3
n
n
u
transcribes genes into mRNA
only transcribes tRNA genes
each has a specific promoter sequence
it recognizes
AP Biology
Which gene is read?
n
Promoter region
binding site before beginning of gene
TATA box binding site
u binding site for RNA polymerase
& transcription
factors
u
u
n
Enhancer region
u
binding site far
upstream of gene
n
turns transcription
on HIGH
AP Biology
4
Division Ave. High School
Ms. Foglia
AP Biology
Transcription Factors
n
Initiation complex
transcription factors bind to promoter region
u
n
n
n
suite of proteins which bind to DNA
hormones?
turn on or off transcription
trigger the binding of RNA polymerase to DNA
u
AP Biology
Matching bases of DNA & RNA
n
Match RNA bases to DNA
bases on one of the DNA
strands
G
A
C
U
A
G
G
U
U
C
A
AG
C
G
A
U
U
C
A
C
5'
RNA
A
U
3'
A C C polymerase G
T GG T A C A G C T A G T C A T CG T A C CG T
AP Biology
Eukaryotic genes have junk!
n
Eukaryotic genes are not continuous
u
exons = the real gene
n
u
expressed / coding DNA
introns
come out!
introns = the junk
n
inbetween sequence
intron = noncoding (inbetween) sequence
eukaryotic DNA
exon = coding (expressed) sequence
AP Biology
5
Division Ave. High School
Ms. Foglia
AP Biology
mRNA splicing
n
Post-transcriptional processing
u
u
u
u
eukaryotic mRNA needs work after transcription
primary transcript = pre-mRNA
mRNA splicing
n edit out introns
make mature mRNA transcript
intron = noncoding (inbetween) sequence
~10,000 bases
eukaryotic DNA
exon = coding (expressed) sequence
primary mRNA
transcript
AP Biology
~1,000 bases
mature mRNA
transcript
spliced mRNA
Discovery of exons/introns
Richard
Roberts
CSHL
AP Biology
pre-mRNA
Philip
Sharp
MIT
1977 | 1993
adenovirus
common cold
beta-thalassemia
Splicing must be accurate
n
No room for mistakes!
u
a single base added or lost throws off the
reading frame
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGUCCGAUAAGGGCCAU
AUG|CGG|UCC|GAU|AAG|GGC|CAU
Met|Arg|Ser|Asp|Lys|Gly|His
AP Biology
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
Met|Arg|Val|Arg|STOP|
6
Division Ave. High School
Ms. Foglia
AP Biology
Whoa! I think
we just broke
a biological “rule”!
RNA splicing enzymes
n
snRNPs
small nuclear RNA
exon
proteins
u
u
n
Spliceosome
intron
u
n
exon
5'
3'
several snRNPs
recognize splice
site sequence
u
snRNPs
snRNA
spliceosome
5'
3'
cut & paste gene
lariat
No,
not smurfs!
“snurps”
5'
mature mRNA
AP Biology
exon
5'
3'
exon
3'
excised
intron
Alternative splicing
n
Alternative mRNAs produced from same gene
u
u
when is an intron not an intron…
different segments treated as exons
Starting to get
hard to
define a gene!
AP Biology
More post-transcriptional processing
n
Need to protect mRNA on its trip from
nucleus to cytoplasm
u
enzymes in cytoplasm attack mRNA
protect the ends of the molecule
add 5¢ GTP cap
n add poly-A tail
n
n
w longer tail, mRNA lasts longer: produces more protein
3'
mRNA
5'
P
G P
A
P
AP Biology
7
Division Ave. High School
Ms. Foglia
AP Biology
a
a
From gene to protein
nucleus
DNA
cytoplasm
transcription
mRNA
a
a
translation
a
a
a
a
a
a
a
a
a
a
a
protein
a
a
a
a
a
a
a
ribosome
trait
AP Biology
Translation
from
nucleic acid language
to
amino acid language
AP Biology
2007-2008
How does mRNA code for proteins?
DNA
TACGCACATTTACGTACGCGG
mRNA
AUGCGUGUAAAUGCAUGCGCC
4 ATCG
4 AUCG
protein
?
Met Arg Val Asn Ala Cys Ala
20
AP Biology
How can you code for 20 amino acids
with only 4 nucleotide bases (A,U,G,C)?
8
Division Ave. High School
Ms. Foglia
AP Biology
mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGG
DNA
codon
AUGCGUGUAAAUGCAUGCGCC
mRNA
?
protein
Met Arg Val Asn Ala Cys Ala
AP Biology
1960 | 1968
Cracking the code
n
Nirenberg & Khorana
Crick
u
determined 3-letter (triplet) codon system
WHYDIDTHEREDBATEATTHEFATRAT
n
Nirenberg (47) & Khorana (17)
u
u
determined mRNA–amino acid match
added fabricated mRNA to test tube of
ribosomes, tRNA & amino acids
n
n
created artificial UUUUU… mRNA
found that UUU coded for phenylalanine
AP Biology
Marshall Nirenberg
1960 | 1968
Har Khorana
AP Biology
9
Division Ave. High School
Ms. Foglia
AP Biology
The code
n
Code for ALL life!
strongest support for
a common origin for
all life
u
n
Code is redundant
several codons for
each amino acid
3rd base “wobble”
u
u
Why is the
wobble good?
Start codon
n
u
u
AUG
methionine
Stop codons
n
AP Biology
u
UGA, UAA, UAG
How are the codons matched to
amino acids?
DNA
mRNA
3¢
5¢
5¢
3¢
TACGCACATTTACGTACGCGG
AUGCGUGUAAAUGCAUGCGCC
3¢
tRNA
amino
acid
UAC
Met
codon
5¢
GCA
Arg
CAU
anti-codon
Val
AP Biology
a
a
From gene to protein
nucleus
DNA
cytoplasm
transcription
mRNA
a
a
translation
a
a
a
a
a
a
a
a
a
a
a
protein
a
a
a
a
a
a
a
ribosome
trait
AP Biology
10
Division Ave. High School
Ms. Foglia
AP Biology
Transfer RNA structure
n
“Clover leaf” structure
u anticodon on “clover leaf” end
u amino acid attached on 3¢ end
AP Biology
Loading tRNA
n
Aminoacyl tRNA synthetase
enzyme which bonds amino acid to tRNA
bond requires energy
u
u
ATP ® AMP
bond is unstable
so it can release amino acid at ribosome easily
n
n
n
Trp C=O
OH
OH
Trp C=O
O
Trp
H2O
O
activating
enzyme
tRNATrp
anticodon
AP Biology
tryptophan attached
to tRNATrp
AC C
UGG
mRNA
tRNATrp binds to UGG
condon of mRNA
Ribosomes
n
Facilitate coupling of
tRNA anticodon to
mRNA codon
n
Structure
u
u
u
organelle or enzyme?
ribosomal RNA (rRNA) & proteins
2 subunits
n
n
large
small
E P A
AP Biology
11
Division Ave. High School
Ms. Foglia
AP Biology
Ribosomes
n
n
n
A site (aminoacyl-tRNA site)
u holds tRNA carrying next amino acid to
be added to chain
P site (peptidyl-tRNA site)
u holds tRNA carrying growing
polypeptide chain
Met
E site (exit site)
u empty tRNA
leaves ribosome
5'
from exit site
U A C
A U G
E
AP Biology
P
3'
A
Building a polypeptide
n
Initiation
u
n
Elongation
u
n
brings together mRNA, ribosome
subunits, initiator tRNA
adding amino acids based on
codon sequence
Termination
u
3 2 1
end codon
Leu
Val
Met
Met
Met
Met Leu
release
factor
Ser
Ala
Leu
Leu
Trp
tRNA
U AC
5'
GC U G A A U
mRNA A U
3'
E
P
A
5'
UAC GAC
A U G C U GA A U
5'
3'
U A C GA C
A U G C U G AA U
3'
5'
U A C G A C AA U
AU G C UG
3'
A CC
U GG U A A
3'
AP Biology
Protein targeting
n
Signal peptide
u
address label
Destinations:
n
n
n
n
n
n
start of a secretory pathway
n
secretion
nucleus
mitochondria
chloroplasts
cell membrane
cytoplasm
etc…
AP Biology
12
Division Ave. High School
Ms. Foglia
AP Biology
RNA polymerase
DNA
Can you tell
the story?
amino
acids
exon
intron
tRNA
pre-mRNA
5' GTP cap
mature mRNA
aminoacyl tRNA
synthetase
poly-A tail
large ribosomal subunit
3'
polypeptide
5'
tRNA
small ribosomal subunit
E P A
AP Biology
ribosome
The Transcriptional unit (gene?)
enhancer
1000+b
3'
translation
start
20-30b
TAC
RNA
TATA
polymerase
DNA
transcriptional unit (gene)
UTR
promoter
translation
stop
exons
UTR
introns
transcription
start
transcription
stop
5'
pre-mRNA
AP Biology
5'
DNA
ACT
3'
5'
GTP mature mRNA
3'
AAAAAAAA
Bacterial chromosome
Protein
Synthesis in
Prokaryotes
Transcription
mRNA
Psssst…
no nucleus!
Cell
membrane
Cell wall
AP Biology
2007-2008
13
Division Ave. High School
Ms. Foglia
AP Biology
Prokaryote vs. Eukaryote genes
n
Prokaryotes
u
DNA in cytoplasm
circular
chromosome
naked DNA
u
no introns
u
u
n
Eukaryotes
u
u
u
u
DNA in nucleus
linear
chromosomes
DNA wound on
histone proteins
introns vs. exons
introns
come out!
intron = noncoding (inbetween) sequence
eukaryotic
DNA
exon = coding (expressed) sequence
AP Biology
Translation in Prokaryotes
n
Transcription & translation are simultaneous
in bacteria
DNA is in
cytoplasm
u no mRNA
editing
u ribosomes
read mRNA
as it is being
transcribed
u
AP Biology
Translation: prokaryotes vs. eukaryotes
n
Differences between prokaryotes &
eukaryotes
u
time & physical separation between
processes
n
u
takes eukaryote ~1 hour
from DNA to protein
no RNA processing
AP Biology
14