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From Gene
to Protein
How Genes
Work
AP Biology
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
AP Biology
to produce a certain amino acid
 non-functional enzyme from damaged gene
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
amino acid p-Amino
Niacin
Inositol acid Folic
supplements
acid
Thiamine
benzoic acid
AP Biology
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
enzyme E
glutamate
arg-G arg-H
arg-F
enzyme F
ornithine
substrate in
AP biochemical
Biology
pathway
enzyme G
citruline
enzyme H
arginosuccinate
arginine
gene that
was damaged
The “Central Dogma”

Flow of genetic information in a cell

How do we move information from DNA to proteins?
DNA
replication
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RNA
protein
DNA gets
all the glory,
but proteins do
all the work!
trait
RNA


ribose sugar
N-bases
uracil instead of thymine
U : A
C : G



single stranded
lots of RNAs

DNA
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mRNA, tRNA, rRNA, siRNA…
transcription
RNA
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


synthesis of complementary RNA strand


same sequence as RNA
transcription bubble
enzyme

RNA polymerase
5
C
DNA
G
3
A
G
T
A T C
T A
53
G
A G C
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
C
A G
C T
G
A
T
A
T
3
5
unwinding
rewinding
mRNA
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build
RNA
coding strand
5
RNA polymerase
template strand
Bacterial chromosome
Transcription
in Prokaryotes
Psssst…
no nucleus!
Cell
membrane
Cell wall
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Transcription
mRNA
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 35
APBiology
build RNA 53

Transcription in Prokaryotes

Promoter sequences
enzyme
subunit
RNA polymerase
read DNA 35
bacterial DNA
Promoter
TTGACA TATAAT
–35 sequence
–10 sequence
RNA polymerase
AP Biology
strong vs. weak promoters
Transcription in Prokaryotes

Elongation

RNA polymerase
copies DNA as it
unwinds


~20 base pairs at a time
 300-500 bases in gene
builds RNA 53
Simple proofreading
1 error/105 bases
 make many mRNAs
 mRNA has short life
not worth editing!
AP 
Biology

reads DNA 35
Transcription in Prokaryotes

Termination

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RNA polymerase stops at termination
sequence
Transcription in Eukaryotes
Transcription
Psssst…
DNA can’t
leave nucleus!
RNA Processing
Translation
Protein
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Prokaryote vs. Eukaryote genes

Prokaryotes

Eukaryotes


DNA in cytoplasm
circular
chromosome
naked DNA

no introns





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
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Transcription in Eukaryotes

3 RNA polymerase enzymes

RNA polymerase 1



RNA polymerase 2


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transcribes genes into mRNA
RNA polymerase 3


only transcribes rRNA genes
makes ribosomes
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
AP Biology
Post-transcriptional processing

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
mRNA
P
 add polyA tail
5' G PP
3'
A
intron = noncoding (inbetween) sequence
~10,000 bases
eukaryotic DNA
exon = coding (expressed) sequence
pre-mRNA
primary mRNA
transcript
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mature mRNA
transcript
~1,000 bases
spliced mRNA
Alteration of Pre-mRNA ends

add 5 cap
Modified guanine nucleotide
 Protects mRNA from enzymes
 Helps ribosome recognize attachment site


add polyA tail
About 30-200 adenine nucleotides added
 Protects RNA from enzymes in cytoplasm
 May facilitate export of mRNA to cytoplasm

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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
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|
Splicing enzymes

snRNPs



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
Ribozyme

RNA as ribozyme
some mRNA can even splice itself
 RNA as enzyme

Sidney Altman
AP Biology
Yale
Thomas Cech
U of Colorado
Translation
from
nucleic acid language
to
amino acid language
AP Biology
Bacterial chromosome
Translation in
Prokaryotes
Transcription
mRNA
Translation
Psssst…
no nucleus!
protein
Cell
membrane
Cell wall
AP Biology
Translation in Prokaryotes

Transcription & translation are simultaneous
in bacteria
DNA is in
cytoplasm
 no mRNA
editing
 ribosomes
read mRNA
as it is being
transcribed
 Smaller
ribosomes

AP Biology
Translation: prokaryotes vs. eukaryotes

Differences between prokaryotes &
eukaryotes

time & physical separation between
processes


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takes eukaryote ~1 hour
from DNA to protein
RNA processing
Translation in Eukaryotes
AP Biology
mRNA codes for proteins in triplets
DNA
TACGCACATTTACGTACGCGG
codon
mRNA
AUGCGUGUAAAUGCAUGCGCC
?
protein
AP Biology
Met Arg Val Asn Ala Cys Ala
Cracking the code

Crick

determined 3-letter (triplet) codon system
WHYDIDTHEREDBATEATTHEFATRAT

Nirenberg & Khorana
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)
Other tests (AAA, GGG…) supported results
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”
Why is the
wobble good?

Start codon



AP Biology
AUG
methionine
Stop codons

UGA, UAA, UAG
Wobble





1 tRNA can recognize 2-3 different mRNA
codons
Third base of a codon is known as the
"wobble position"
Explains why the multiple codons per
amino acid differ by the third position
45 types of tRNA  translate 64 codons
most versatile tRNA's are those with
Inosine (I), a modified base, in the wobble
position

AP Biology
I can hydrogen bond with U, C, or A.
How are the codons matched to
amino acids?
DNA
mRNA
3
5
TACGCACATTTACGTACGCGG
AUGCGUGUAAAUGCAUGCGCC
codon
3
tRNA
UAC
amino
acid
Met
AP Biology
5
GCA
Arg
CAU
Val
anti-codon
5
3
Transfer RNA structure

“Clover leaf” structure
anticodon on “clover leaf” end
 amino acid attached on 3 end

AP Biology
Loading tRNA

Aminoacyl tRNA synthetase


Specific enzyme which bonds amino acid to specific
tRNA
bond requires energy


ATP  AMP
energy stored in tRNA-amino acid bond

Unstable = so it can release amino acid at ribosome easily
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

Facilitate coupling of
tRNA anticodon to
mRNA codon

Structure




ribosomal RNA (rRNA) & proteins
Made in nucleolus
Assemble when attached to mRNA
2 subunits


large
small
E P A
Ribosomes

A site (aminoacyl-tRNA site)


P site (peptidyl-tRNA site)


holds tRNA carrying next amino acid to
be added to chain
holds tRNA carrying growing
polypeptide chain
Met
E site (exit site)

AP Biology
empty tRNA
leaves ribosome
from exit site
U A C
A U G
5'
E
P
A
3'
Building a polypeptide



Initiation
 brings together mRNA, ribosome
subunits, initiator tRNA-Met
 AUG = start codon
Elongation
 adding amino acids based on
codon sequence
 Growing polypeptide = peptide
bonds
Termination
 end codon: UAA, UAG, UGA
3 2 1
Leu
Val
Met
Met
Met
Met Leu
Ala
Leu
Leu
release
factor
Ser
Trp
tRNA
U AC
5'
C UGAA U
mRNA A U G
3'
E P A
5'
UAC GAC
A U G C U GAA U
5'
3'
U A C GA C
A U G C U G AAU
5'
3'
U AC G A C
AA U
AU G C U G
3'
A CC
U GG U A A
3'
Polyribosome

A cluster of ribosomes
simultaneously
translating an mRNA
strand
AP Biology
Destinations:
Protein targeting



Signal peptide

address label




start of a secretory pathway
AP Biology

secretion
nucleus
mitochondria
chloroplasts
cell membrane
cytoplasm
etc…
RNA polymerase
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
tRNA
E P A
ribosome
3'
Got Questions?
Can I translate that for you?
AP Biology
Substitute Slides
for Student Print version
AP Biology
Can you tell
the story?
AP Biology
Extra Slides
(used some years & not others)
AP Biology
Translation

Codons

AP Biology
blocks of 3
nucleotides
decoded into
the sequence
of amino acids
Building Proteins

Organelles involved
nucleus
 ribosomes
 endoplasmic reticulum
(ER)
 Golgi apparatus
 vesicles

The Protein Assembly Line
nucleus
AP Biology
ribosome
ER
Golgi
apparatus
vesicles
From nucleus to cytoplasm…

Where are the genes?


Where are proteins synthesized?


genes are on chromosomes in nucleus
proteins made in cytoplasm by ribosomes
How does the information get from DNA
in nucleus to cytoplasm?

AP Biology
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 get
hard to
define a gene!
AP Biology
Domains

Modular architecture
of many proteins


exons may represent
functional units of
protein
easier to mix and match
in the production of new
proteins?
AP Biology
So… What is a gene?

One gene – one enzyme?



One gene – one protein?



but many proteins are composed of several polypeptides
but each polypeptide has its own gene
One gene – one polypeptide?


but not all proteins are enzymes
but all proteins are coded by genes
but many genes only code for RNA (tRNA, rRNA…)
One gene – one product?

AP Biology
but many genes code for
more than one product …
So…
Where does
that leave
us?!
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
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.
TACGCACATTTACGTACGCGGATGCCGCGACTATGATC
ACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACT
human genome
CGACTAGCATGATCGATCAGCTACATGCTAGCACACYC
GTACATCGATCCTGACATCGACCTGCTCGTACATGCTA
3.2
billion
bases
CTAGCTACTGACTCATGATCCAGATCACTGAAACCCTA
GATCGGGTACCTATTACAGTACGATCATCCGATCAGAT
CATGCTAGTACATCGATCGATACTGCTACTGATCTAGC
TCAATCAAACTCTTTTTGCATCATGATACTAGACTAGC
TGACTGATCATGACTCTGATCCCGTAGATCGGGTACCT
ATTACAGTACGATCATCCGATCAGATCATGCTAGTACA
TCGATCGATACTGCTACTGATCTAGCTCAATCAAACTC
TTTTTGCATCATGATACTAGACTAGCTGACTGATCATG
ACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGA
TCATCCGATCAGATCATGCTAGTACATCGATCGATACT
AP Biology
Any Questions??
What color would a smurf turn
if he held his breath?
AP Biology
The Transcriptional unit
enhancer
exons
1000+b
20-30b
3'
RNA
TATA
polymerase
TAC
transcriptional unit
5'
DNA
ACT
introns
5'
3'
5'
AP Biology
3'