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AP Biology
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 to produce a certain amino acid
non-functional enzyme from damaged gene
AP Biology
Wild-typeNeurospora
Minimalmedium
Select one ofthe spores
Grow oncomplete medium
Minimalcontrol
Nucleicacid
CholinePyridoxine Riboflavin Arginine
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
experimentalsmutatio
n identifi
ed
amino acidsupplements
AP Biology
One gene / one enzyme hypothesis
chromosome
genecluster 1
enzyme E
glutamate ornithine citruline argino-succinate
arginine
enzyme F enzyme G enzyme H
encodedenzyme
substrate inbiochemical pathway
genecluster 2
genecluster 3
arg-Harg-Garg-Farg-E
Damage to specific gene, mapped to nutritional mutations
gene thatwas damaged
AP Biology
The “Central Dogma” Flow of genetic information in a cell
How do we move information from DNA to proteins?
transcriptiontranslation
replication
proteinRNADNA trait
DNA gets all the glory,
but proteins do all the work!
AP Biology
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
Transcription
fromDNA nucleic acid language
toRNA nucleic acid language
AP Biology
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
mRNA RNA polymerase
unwinding
coding strand
DNAC C
C
C
C
C
C
C
C CC
G
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
AP Biology
Transcription in Prokaryotes
Bacterial chromosome
mRNA
Cell wall
Cellmembrane
Transcription
Psssst…no nucleus!
AP Biology
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
AP Biology
Transcription in Prokaryotes Promoter sequences
TATAAT
RNA polymerase
Promoter
enzymesubunit
bacterial DNA
–35 sequence –10 sequenceTTGACA
RNA polymerasestrong vs. weak promoters
read DNA 35
AP Biology
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
AP Biology
Transcription in Prokaryotes Termination
RNA polymerase stops at termination sequence
AP Biology
Transcription in Eukaryotes
Protein
RNA Processing
Translation
Transcription
Psssst…DNA can’t
leave nucleus!
AP Biology
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
eukaryoticDNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
intronscome out!
AP Biology
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
AP Biology
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
A A A A A3' poly-A tail
mRNA
5'5' cap
3'
GPPP
50-250 A’s
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
AP Biology
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
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
AUG|CGG|UCC|GAU|AAG|GGC|CAU
AUGCGGCTATGGGUCCGAUAAGGGCCAUAUGCGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
AUGCGGCTATGGGUCCGAUAAGGGCCAUAUGCGGGUCCGAUAAGGGCCAU
Met|Arg|Ser|Asp|Lys|Gly|His
Met|Arg|Val|Arg|STOP|
AP Biology
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
AP Biology
Ribozyme
Sidney Altman Thomas CechYale U of Colorado
RNA as ribozyme some mRNA can even splice itself RNA as enzyme
AP Biology
Translation
fromnucleic acid language
toamino acid language
AP Biology
Translation in Prokaryotes
Bacterial chromosome
mRNA
Cell wall
Cellmembrane
Transcription
Translation
proteinPsssst…no nucleus!
AP Biology
Transcription & translation are simultaneous in bacteria DNA is in
cytoplasm no mRNA
editing ribosomes
read mRNA as it is being transcribed
Smaller ribosomes
Translation in Prokaryotes
AP Biology
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
AP Biology
AUGCGUGUAAAUGCAUGCGCCmRNA
mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
Met Arg Val Asn Ala Cys Alaprotein
?
codon
AP Biology
Cracking the code Crick
determined 3-letter (triplet) codon system
WHYDIDTHEREDBATEATTHEFATRATWHYDIDTHEREDBATEATTHEFATRAT
Nirenberg & Khorana 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) Other tests (AAA, GGG…) supported results
AP Biology
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?
AP Biology
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 I can hydrogen bond with U, C, or A.
AP Biology
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
AP Biology
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
activatingenzyme
anticodontRNATrp binds to UGG
condon of mRNA
Trp Trp Trp
mRNAA C CU GG
C=O
OHOH
H2OO
tRNATrp
tryptophan attached to tRNATrp
C=O
O
C=O
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
AP Biology
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-Met
AUG = start codon Elongation
adding amino acids based on codon sequence
Growing polypeptide = peptide bonds
Termination end codon: UAA, UAG, UGA
123
Leu
Leu Leu Leu
tRNA
Met MetMet Met
PE AmRNA5' 5' 5' 5'
3' 3' 3'3'
U UA AAACC
CAU UG G
GUU
A AAAC
CC
AU UG GGU
UA
AAAC
CC
AU UG GGU U
A AACCA U UG G
G AC
ValSer
AlaTrp
releasefactor
AA A
CCU UGG 3'
AP Biology
Polyribosome A cluster of ribosomes
simultaneously translating an mRNA strand
AP Biology
Protein targeting Signal peptide
address label
Destinations: secretion nucleus mitochondria chloroplasts cell membrane cytoplasm etc…start of a secretory pathway
AP Biology
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
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
blocks of 3 nucleotides decoded into the sequence of amino acids
AP Biology
Building Proteins Organelles involved
nucleus ribosomes endoplasmic reticulum
(ER) Golgi apparatus vesicles
nucleus ribosome ERGolgi
apparatusvesicles
The Protein Assembly Line
AP Biology
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
AP Biology
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!
AP Biology
Domains
Modular architectureof 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?
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?!
AP Biology
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 know
what a wabbitlooks like.
AP Biology
TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACT
human genome3.2 billion bases
AP Biology
Any Questions??
What color would a smurf turnif he held his breath?
AP Biology
20-30b
3'
introns
The Transcriptional unit
transcriptional unitTAC ACTDNATATA
5'RNApolymerase
5' 3'
5' 3'
exonsenhancer
1000+b
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