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From Gene to Protein

How Genes Work

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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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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

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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

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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!

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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…

RNADNAtranscription

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Transcription

fromDNA nucleic acid language

toRNA 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

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

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Transcription in Prokaryotes

Bacterial chromosome

mRNA

Cell wall

Cellmembrane

Transcription

Psssst…no nucleus!

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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 35 build RNA 53

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Transcription in Prokaryotes Promoter sequences

TATAAT

RNA polymerase

Promoter

enzymesubunit

bacterial DNA

–35 sequence –10 sequenceTTGACA

RNA polymerasestrong vs. weak promoters

read DNA 35

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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

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Transcription in Prokaryotes Termination

RNA polymerase stops at termination sequence

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Transcription in Eukaryotes

Protein

RNA Processing

Translation

Transcription

Psssst…DNA can’t

leave nucleus!

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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

eukaryoticDNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

intronscome out!

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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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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

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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

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|

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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

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Ribozyme

Sidney Altman Thomas CechYale U of Colorado

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

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Translation

fromnucleic acid language

toamino acid language

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Translation in Prokaryotes

Bacterial chromosome

mRNA

Cell wall

Cellmembrane

Transcription

Translation

proteinPsssst…no nucleus!

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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

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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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AUGCGUGUAAAUGCAUGCGCCmRNA

mRNA codes for proteins in triplets

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

Met Arg Val Asn Ala Cys Alaprotein

?

codon

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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

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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”

Start codon AUG methionine

Stop codons UGA, UAA, UAG

Why is thewobble good?

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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.

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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

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Transfer RNA structure “Clover leaf” structure

anticodon on “clover leaf” end amino acid attached on 3 end

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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

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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'

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Polyribosome A cluster of ribosomes

simultaneously translating an mRNA strand

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Protein targeting Signal peptide

address label

Destinations: secretion nucleus mitochondria chloroplasts cell membrane cytoplasm etc…start of a secretory pathway

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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

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Got Questions? Can I translate that for you?

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Substitute Slides for Student Print version

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Can you tell the story?

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Extra Slides (used some years & not others)

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Translation Codons

blocks of 3 nucleotides decoded into the sequence of amino acids

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Building Proteins Organelles involved

nucleus ribosomes endoplasmic reticulum

(ER) Golgi apparatus vesicles

nucleus ribosome ERGolgi

apparatusvesicles

The Protein Assembly Line

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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

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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!

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Domains

Modular architectureof many proteins exons may represent

functional units of protein

easier to mix and match in the production of new proteins?

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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?!

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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.

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TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACT

human genome3.2 billion bases

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Any Questions??

What color would a smurf turnif he held his breath?

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20-30b

3'

introns

The Transcriptional unit

transcriptional unitTAC ACTDNATATA

5'RNApolymerase

5' 3'

5' 3'

exonsenhancer

1000+b