AP

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From Gene to Protein
How Genes Work
2
Making proteins

• Organelles
• nucleus
• ribosomes
• endoplasmic reticulum (ER)
• Golgi apparatus
• vesicles

small ribosomal subunit
nuclear pore
mRNA
large ribosomal subunit
cytoplasm
3
Nucleus Nucleolus
4
Nucleolus

• Function
• ribosome production
• build ribosome subunits from rRNA proteins
• exit through nuclear pores to cytoplasm
  combine to form functional ribosomes

rRNA proteins
nucleolus
5
Ribosomes

• Function
• protein production
• Structure
• rRNA protein
• 2 subunits combine

6
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

membrane proteins
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endoplasmicreticulum
nucleus
proteinon its way!
DNA
RNA
vesicle
vesicle
ribosomes
TO
protein
finishedprotein
Golgi apparatus
Making Proteins
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TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGC
TGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTAC
ATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGC
TACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTAC
CTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATA
CTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGAC
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?
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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

Am I just the sum of my proteins?
metabolic pathway
?
?
?
?
enzyme 1
enzyme 2
enzyme 3
enzyme 4
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ingested protein
digestion
phenylalanine
phenylalanine hydroxylase
tyrosine
melanin
thyroxine
transaminase
hydroxyphenylpyruvicacid
hydroxyphenylpyruvic acidoxidase
homogentisicacid
homogentisic acidoxidase
maleylacetoaceticacid
CO2 H2O
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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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Beadle Tatum
create mutations
positive control
negative control
mutation identified
experimentals
amino acidsupplements
13
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
arg-H
arg-G
arg-F
encoded enzyme
enzyme E
enzyme F
enzyme G
enzyme H
glutamate
ornithine
citruline
arginine
argino- succinate
gene thatwas damaged
substrate in biochemical pathway
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Beadle Tatum
1941 1958
one gene one enzyme hypothesis
George Beadle
Edward Tatum
"for their discovery that genes act by regulating
definite chemical events"
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The Central Dogma

• Flow of genetic information in a cell
• How do we move information from DNA to proteins?

transcription
translation
protein
RNA
DNA
trait
DNA gets all the glory, but proteins do all
the work!
replication
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RNA

• ribose sugar
• N-bases
• uracil instead of thymine
• U A
• C G
• single stranded
• lots of RNAs
• mRNA, tRNA, rRNA, siRNA

transcription
RNA
DNA
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Transcription

• fromDNA nucleic acid languagetoRNA 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

coding strand
3?
C
C
G
G
A
A
T
T
5?
A
G
A
A
A
C
G
T
T
T
T
C
A
T
C
G
C
A
T
DNA
3?
C
T
G
A
A
5?
T
G
C
C
G
G
A
U
U
T
C
unwinding
3?
C
G
G
A
A
T
rewinding
mRNA
template strand
RNA polymerase
5?
build RNA 5??3?
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Transcription in Prokaryotes
Bacterial chromosome
Transcription
mRNA
Psssstno nucleus!
Cell membrane
Cell wall
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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?
• build RNA 5??3?

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Transcription in Prokaryotes

• Promoter sequences

enzymesubunit
RNA polymerase
read DNA 3??5?
bacterial DNA
Promoter
TATAAT
TTGACA
35 sequence
10 sequence
RNA polymerase molecules bound to bacterial DNA
RNA polymerase
strong vs. weak promoters
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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!

reads DNA 3??5?
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Transcription in Prokaryotes

• Termination
• RNA polymerase stops at termination sequence

RNA GC hairpin turn
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Transcription in Eukaryotes
Transcription
RNA Processing
PsssstDNA cantleave nucleus!
Translation
Protein
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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

intronscome out!
eukaryotic DNA
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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

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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

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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
• add polyA tail

3' poly-A tail
3'
A
A
A
A
A
mRNA
50-250 As
5' cap
P
P
P
5'
G
10,000 bases
eukaryotic DNA
pre-mRNA
primary mRNA transcript
1,000 bases
mature mRNA transcript
spliced mRNA
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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
AUGCGGUCCGAUAAGGGCCAU
MetArgSerAspLysGlyHis
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
MetArgValArgSTOP
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Splicing enzymes
Whoa! I think we just brokea biological rule!

• snRNPs
• small nuclear RNA
• proteins
• Spliceosome
• several snRNPs
• recognize splice site sequence
• cut paste

No, not smurfs! snurps
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Ribozyme
1982 1989

• RNA as ribozyme
• some mRNA can even splice itself
• RNA as enzyme

Sidney Altman
Thomas Cech
Yale
U of Colorado
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Translation

• fromnucleic acid languagetoamino acid language

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Translation

• Codons
• blocks of 3 nucleotides decoded into the
  sequence of amino acids

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Translation in Prokaryotes
Bacterial chromosome
Transcription
mRNA
Translation
Psssstno nucleus!
protein
Cell membrane
Cell wall
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Translation in Prokaryotes

• Transcription translation are simultaneous in
  bacteria
• DNA is in cytoplasm
• no mRNA editing
• ribosomes read mRNA as it is being transcribed
  

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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

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Translation in Eukaryotes
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From gene to protein
transcription
translation
mRNA
DNA
protein
mRNA leaves nucleus through nuclear pores
proteins synthesized by ribosomes using
instructions on mRNA
nucleus
cytoplasm
39
How does mRNA code for proteins?
4
ATCG
4
AUCG
20

• How can you code for 20 amino acids with only 4
  nucleotide bases (A,U,G,C)?

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mRNA codes for proteins in triplets
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Cracking the code
1960 1968
Nirenberg Khorana

• Crick
• determined 3-letter (triplet) codon system

WHYDIDTHEREDBATEATTHEFATRAT
WHYDIDTHEREDBATEATTHEFATRAT

• Nirenberg (47) Khorana (17)
• determined mRNAamino 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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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 thewobble good?

• Start codon
• AUG
• methionine
• Stop codons
• UGA, UAA, UAG

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How are the codons matched to amino acids?
3?
5?
TACGCACATTTACGTACGCGG
DNA
5?
3?
AUGCGUGUAAAUGCAUGCGCC
mRNA
codon
3?
5?
tRNA
aminoacid
anti-codon
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From gene to protein
transcription
translation
mRNA
DNA
protein
nucleus
cytoplasm
45
Transfer RNA structure

• Clover leaf structure
• anticodon on clover leaf end
• amino acid attached on 3? end

46
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

Trp
CO
Trp
Trp
CO
H2O
OH
O
OH
CO
O
activating enzyme
tRNATrp
A
C
C
mRNA
U
G
G
anticodon
tryptophan attached to tRNATrp
tRNATrp binds to UGG condon of mRNA
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Ribosomes

• Facilitate coupling of tRNA anticodon to mRNA
  codon
• organelle or enzyme?
• Structure
• ribosomal RNA (rRNA) proteins
• 2 subunits
• large
• small

E
P
A
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Ribosomes

• 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

Met
C
A
U
5'
G
U
A
3'
A
P
E
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Building a polypeptide

• Initiation
• brings together mRNA, ribosome subunits,
  initiator tRNA
• Elongation
• adding amino acids based on codon sequence
• Termination
• end codon

release factor
Leu
Val
Ser
Met
Met
Ala
Leu
Met
Met
Leu
Leu
Trp
tRNA
C
A
G
C
A
G
C
C
A
A
C
G
U
A
C
G
C
A
C
U
A
U
A
U
U
A
5'
5'
A
A
A
G
5'
U
C
U
A
5'
G
G
A
A
A
G
U
U
U
C
U
G
G
U
U
3'
C
A
U
C
G
G
A
U
A
U
A
A
C
C
mRNA
3'
3'
3'
A
A
U
U
G
G
3'
P
E
A
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Protein targeting

• Destinations
• secretion
• nucleus
• mitochondria
• chloroplasts
• cell membrane
• cytoplasm
• etc

• Signal peptide
• address label

start of a secretory pathway
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RNA polymerase
DNA
Can you tell the story?
aminoacids
exon
intron
tRNA
pre-mRNA
5' cap
mature mRNA
aminoacyl tRNAsynthetase
polyA tail
3'
large ribosomal subunit
polypeptide
5'
tRNA
small ribosomal subunit
E
P
A
ribosome
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Got Questions? Can I translate that for you?