click to add text ap biology 2007-2008 from gene to protein how genes work

2007-2008 AP Biology

From Gene to Protein

How Genes Work

AP Biology

Organelles nucleus ribosomes endoplasmic reticulum

(ER) Golgi apparatus vesicles

Making proteins

smallribosomal

subunit

largeribosomal

subunit

cytoplasm

mRNA

nuclear pore

AP Biology

Nucleus & Nucleolus

AP Biology

Nucleolus Function

ribosome production build ribosome subunits from rRNA & proteins exit through nuclear pores to cytoplasm &

combine to form functional ribosomes

smallsubunit

large subunit

ribosome

rRNA &proteins

nucleolus

AP Biology

smallsubunit

largesubunitRibosomes

Function protein production

Structure rRNA & protein 2 subunits combine 0.08m

RibosomesRough

ER

SmoothER

AP Biology membrane proteins

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

AP Biology

DNA

RNA

ribosomes

endoplasmicreticulum

vesicle

Golgi apparatus

vesicle

proteinon its way!

protein finishedprotein

Making Proteins

TO:

TO:

TO:

TO:nucleus

TO:

AP Biology

TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTA

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?

AP Biology

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?

Metabolism taught us about genes

A B C D E

disease disease disease disease

enzyme 1 enzyme 2 enzyme 3 enzyme 4

metabolic pathway

AP Biology

ingested protein

phenylalanine

tyrosine

hydroxyphenylpyruvicacid

homogentisicacid

maleylacetoaceticacid

CO2 & H2O

phenylalanine hydroxylase

transaminase

hydroxyphenylpyruvic acidoxidase

homogentisic acidoxidase

melanin

thyroxine

PKUphenylketonuria

tyrosinosis

alkaptonuria

albinism

cretinism

digestion

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

Beadle & Tatum 1941 | 1958

George Beadle

Edward Tatum

"for their discovery that genes act by regulating definite chemical events"

one gene : one enzyme hypothesis

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


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


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

RNA polymerase molecules bound to bacterial DNA

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

RNA GC hairpin turn


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'

G PPP

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

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

Whoa! I think we just broke

a biological “rule”!

AP Biology

Ribozyme

Sidney Altman Thomas Cech

1982 | 1989

Yale U of Colorado

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


Translation

fromnucleic acid language

toamino acid language

AP Biology

Translation Codons

blocks of 3 nucleotides decoded into the sequence of amino acids


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

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


Translation in Eukaryotes

AP Biology

mRNA

From gene to protein

DNAtranscription

nucleus cytoplasm

mRNA leaves nucleus through nuclear pores

proteins synthesized by ribosomes using instructions on mRNA

aa

aa

aa

aa

aa

aa

aa

aa

ribosome

proteintranslation

AP Biology

How does mRNA code for proteins?

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

Met Arg Val Asn Ala Cys Alaprotein

?

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

4

4

20

ATCG

AUCG

AP Biology


mRNA codes for proteins in triplets



Met Arg Val Asn Ala Cys Alaprotein

?

codon

AP Biology

Cracking the code1960 | 1968

Crick determined 3-letter (triplet) codon system

Nirenberg & Khorana

WHYDIDTHEREDBATEATTHEFATRATWHYDIDTHEREDBATEATTHEFATRAT

Nirenberg (47) & Khorana (17) 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)

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

How are the codons matched to amino acids?



aminoacid

tRNA

anti-codon

codon

5 3

3 5

3 5

UAC

MetGCA

ArgCAU

Val

AP Biology

mRNA

From gene to protein

DNAtranscription

nucleuscytoplasm

aa

aa

aa

aa

aa

aa

aa

aa

ribosome

proteintranslation

aa

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

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

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

AP Biology

Ribosomes Facilitate coupling of

tRNA anticodon to mRNA codon organelle or enzyme?

Structure ribosomal RNA (rRNA) & proteins 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

AP Biology

Building a polypeptide Initiation

brings together mRNA, ribosome subunits, initiator tRNA

Elongation adding amino acids based on

codon sequence

Termination end codon 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

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


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