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AP Biology

AP Biology 2017

How Genes

Work

Organelles

nucleus

ribosomes

endoplasmic reticulum (ER)

Golgi apparatus

vesicles

Making proteins

smallribosomalsubunit

largeribosomalsubunit

cytoplasm

mRNA

nuclear pore

Nucleus & NucleolusNucleolus

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

smallsubunit

largesubunitRibosomes

Function

protein production

Structure

rRNA & protein

2 subunits combine 0.08mm

Ribosomes

RoughER

SmoothER

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

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DNA

RNA

ribosomes

endoplasmic

reticulum

vesicle

Golgi

apparatus

vesicle

proteinon its way!

protein finishedprotein

Making Proteins

TO:

TO:

TO:

TO:

nucleus

TO:

End of the Tour

TACGCACATTTACGTACGCGGATGCCGCGA

CTATGATCACATAGACATGCTGTCAGCTCT

AGTAGACTAGCTGACTCGACTAGCATGATC

GATCAGCTACATGCTAGCACACYCGTACAT

CGATCCTGACATCGACCTGCTCGTACATGC

TACTAGCTACTGACTCATGATCCAGATCAC

TGAAACCCTAGATCGGGTACCTATTACAGT

ACGATCATCCGATCAGATCATGCTAGTACA

TCGATCGATACTGCTACTGATCTAGCTCAA

TCAAACTCTTTTTGCATCATGATACTAGAC

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?

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

ingested protein

phenylalanine

tyrosine

hydroxyphenylpyruvicacid

homogentisicacid

maleylacetoaceticacid

CO2 & H2O

phenylalanine hydroxylase

transaminase

hydroxyphenylpyruvic acidoxidase

homogentisic acidoxidase

melanin

thyroxine

PKUphenylketonuria

tyrosinosis

alkaptonuria

albinism

cretinism

digestion

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 RiboflavinArginine

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

experimentals

amino acidsupplements

One gene / one enzyme hypothesis

chromosome

genecluster 1

enzyme E

glutamate ornithine citruline argino-succinate

arginine

enzyme F enzyme G enzyme H

encoded

enzyme

substrate inbiochemical pathway

genecluster 2

genecluster 3

arg-Harg-Garg-Farg-E

Damage to specific gene, mapped to

nutritional mutations

gene thatwas damaged

Beadle & Tatum 1941 | 1958

George Beadle

Edward Tatum

"for their discovery that genes act by

regulating definite chemical events"

one gene : one enzyme hypothesis

The “Central Dogma”

Flow of genetic information in a cell

How do we move information from DNA to proteins?

replication

proteinRNADNA trait

DNA gets all the glory,

but proteins do all the work!

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 2017

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 same sequence as RNA

synthesis of complementary RNA strand transcription bubble

enzyme RNA polymerase

template strand

rewinding

mRNARNA polymerase

unwinding

coding strand

DNAC C

C

C

C

C

C

C

C CC

G

GG

G

G G

G G

G

G

GA

A

AA A

A

A

A

A

A A

A

AT

T T

T

T

T

T

T

T T

T

T

U U

5

3

5

3

3

5build RNA 53 AP Biology 2017

Transcription

in Prokaryotes

Bacterial chromosome

mRNA

Cell wall

Cellmembrane

Transcription

Psssst…no nucleus!

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

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

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

Transcription in Prokaryotes

Termination

RNA polymerase stops at termination sequence

RNA GC

hairpin turn

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Transcription in Eukaryotes

Protein

RNA Processing

Translation

Transcription

Psssst…DNA can’t

leave nucleus!

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

eukaryotic

DNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

intronscome out!

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

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

AmRNA

5'

3'

GPPP

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

1977 | 1993

Richard Roberts Philip

SharpCSHL

MITadenovirus

common cold

Discovery of Split genes

beta-thalassemia

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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

AUGCGGCTATGGGUCCGAUAAGGGCCAU

AUGCGGUCCGAUAAGGGCCAU

AUG|CGG|GUC|CGA|UAA|GGG|CCA|U

AUGCGGCTATGGGUCCGAUAAGGGCCAU

AUGCGGGUCCGAUAAGGGCCAU

Met|Arg|Ser|Asp|Lys|Gly|His

Met|Arg|Val|Arg|STOP|

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”!

Ribozyme

Sidney Altman Thomas Cech

1982 | 1989

Yale U of Colorado

RNA as ribozyme

some mRNA can even splice itself

RNA as enzyme

AP Biology

Translation

from

nucleic acid language

to

amino acid language

AP Biology

Translation in

Prokaryotes

Bacterial chromosome

mRNA

Cell wall

Cellmembrane

Transcription

Translation

proteinPsssst…no nucleus!

Transcription & translation are simultaneous

in bacteria

DNA is in

cytoplasm

no mRNA

editing

ribosomes

read mRNA

as it is being

transcribed

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

mRNA

From gene to protein

DNAtranscription

nucleus cytoplasm

mRNA leaves nucleus through nuclear pores

proteins synthesized by ribosomes using instructions on mRNA

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

ribosome

proteintranslation

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

AUGCGUGUAAAUGCAUGCGCCmRNA

mRNA codes for proteins in triplets

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

Met Arg Val Asn Ala Cys Alaprotein

?

codon

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)

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1960 | 1968Marshall Nirenberg

Har Khorana

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?

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

mRNA

From gene to protein

DNAtranscription

nucleuscytoplasm

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

ribosome

proteintranslation

aa

Transfer RNA structure

“Clover leaf” structure

anticodon on “clover leaf” end

amino acid attached on 3 end

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

anticodon

tRNATrp binds to UGG condon of mRNA

Trp Trp Trp

mRNAA C CUGG

C=O

OHOH

H2OO

tRNATrp

tryptophan attached to tRNATrp

C=O

O

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Ribosomes

Facilitate coupling of

tRNA anticodon to

mRNA codon

organelle or enzyme?

Structure

ribosomal RNA (rRNA) & proteins

2 subunits

large

smallE P A

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

Elongation adding amino acids based on

codon sequence

Termination end codon 123

Leu

Leu Leu Leu

tRNA

Met MetMet Met

PE A

mRNA5' 5' 5' 5'

3' 3' 3'3'

U UA AAAC

CC

AU UG GGU

UA

AAAC

CC

AU UG GG

UU

AAA

ACC

C

AU UG GGU U

AAAC

CA U UG G

ValSer

AlaTrp

releasefactor

AA A

CCU UGG 3'

Protein targeting

Signal peptide

address label

Destinations: secretion

nucleus

mitochondria

chloroplasts

cell membrane

cytoplasm

etc…start of a secretory pathway

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 2007-2008

Got Questions?

Can I translate that for you?

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COLD STORAGE

Old Slides

Translation

Codons

blocks of 3

nucleotides

decoded into

the sequence

of amino acids

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

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!

Domains

Modular architecture

of many proteins

exons may represent

functional units of

protein

easier to mix and match

in the production of new

proteins?

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 knowwhat a wabbit

looks like.

TACGCACATTTACGTACGCGGATGCCGCGACTATGATC

ACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACT

CGACTAGCATGATCGATCAGCTACATGCTAGCACACYC

GTACATCGATCCTGACATCGACCTGCTCGTACATGCTA

CTAGCTACTGACTCATGATCCAGATCACTGAAACCCTA

GATCGGGTACCTATTACAGTACGATCATCCGATCAGAT

CATGCTAGTACATCGATCGATACTGCTACTGATCTAGC

TCAATCAAACTCTTTTTGCATCATGATACTAGACTAGC

TGACTGATCATGACTCTGATCCCGTAGATCGGGTACCT

ATTACAGTACGATCATCCGATCAGATCATGCTAGTACA

TCGATCGATACTGCTACTGATCTAGCTCAATCAAACTC

TTTTTGCATCATGATACTAGACTAGCTGACTGATCATG

ACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGA

TCATCCGATCAGATCATGCTAGTACATCGATCGATACT

human genome

3.2 billion bases

AAAAAAAAGTP

20-30b

3'

promoter transcription

stop

transcription

start

introns

The Transcriptional unit (gene?)

transcriptional unitTAC ACT

DNA

DNATATA5'RNA

polymerase

pre-mRNA

5' 3'

translation

start

translation

stop

mature mRNA

5' 3'

UTR UTR

exonsenhancer

1000+b20-30b

3'

introns

The Transcriptional unit

transcriptional unitTAC ACT

DNATATA5'RNA

polymerase

5' 3'

5' 3'

exonsenhancer

1000+b