1

From Gene to Phenotype- part 2DNAmolecule

Gene 1

Gene 2

Gene 3

DNA strand(template)

TRANSCRIPTION

mRNA

Protein

TRANSLATION

Amino acid

A C C A A A C C G A G T

U G G U U U G G C U C A

Trp Phe Gly Ser

Codon

3′ 5′

3′5′

Lecture Outline 11/7/05

• The central dogma:– DNA->RNA->protein

• Various types of RNA• The genetic code• Mechanisms of translation

– Initiation, Elongation,Termination

Exam 3 is next Monday. It will cover mitosis andmeiosis, DNA synthesis, transcription, translation,genetics of viruses.(chapters 12, 13, 16, 17, part of 18)

Four types of RNA• mRNA

– Messenger RNA, encodes the amino acid sequence of apolypeptide

• rRNA– Ribosomal RNA, forms complexes with protein called ribosomes,

which translate mRNA to protein• tRNA

– Transfer RNA, transports amino acids to ribosomes during proteinsynthesis

• snRNA– Small nuclear RNA, forms complexes with proteins used in

eukaryotic RNA processing

3′ACCACGCUUAAGACACCU

GC *

GUGUCU

GAGGU

A

A A GUC

AGACC

CGAGA G GG

GACUCAUUUAGGCG5′

Hydrogenbonds

Anticodon

*

*

**

*

**

*

* **

RNA will fold to specificshapes

• Because RNA is single-stranded,parts of the molecule can base pairwith other parts of the samemolecule, causing it to fold intodefined shapes.

• Some RNA molecules can even actas enzymes (ribozymes)

2

Correspondence between exons andprotein domains

GeneDNA

Exon 1 Intron Exon 2 Intron Exon 3

Transcription

RNA processing

Translation

Domain 3

Domain 1

Domain 2

Polypeptide

The genetic codeSecond mRNA base

U C A G

U

C

A

G

UUU

UUCUUA

UUG

CUU

CUC

CUA

CUG

AUU

AUC

AUA

AUG

GUU

GUC

GUA

GUG

Met orstart

Phe

Leu

Leu

lle

Val

UCU

UCCUCA

UCG

CCU

CCC

CCA

CCG

ACU

ACC

ACA

ACG

GCU

GCC

GCA

GCG

Ser

Pro

Thr

Ala

UAU

UAC

UGU

UGCTyr Cys

CAU

CAC

CAA

CAG

CGU

CGC

CGA

CGG

AAU

AAC

AAA

AAG

AGU

AGCAGA

AGG

GAU

GAC

GAA

GAG

GGU

GGC

GGA

GGG

UGG

UAA

UAG Stop

Stop UGA Stop

Trp

His

Gln

Asn

Lys

Asp

Arg

Ser

Arg

Gly

U

CA

G

UC

AG

U

CA

G

UC

AG

Firs

t mR

NA

bas

e (5′ e

nd)

Third

mR

NA

bas

e (3′ e

nd)

Glu

The code is a triplet code– the new boy saw the big cat eat the hot dog

• The first evidence for triplet code came fromdeletion experiments– the new boy saw the big cat eat the hot dog– the neb oys awt heb igc ate att heh otd og

• Function could be restored by an insertionnearby– the new boy saw the big cat eat the hot dog– the neb oys aaw the big cat eat the hot dog

The code is a triplet code

• . . . or by two more deletions to get it backinto the correct “reading frame”– the new boy saw the big cat eat the hot dog– the neb oys awt heb igc ate att heh otd og– the neb osa wte big cat eat the hot dog

3

Deciphering the Code

• Your book tells how people used synthetic mRNAsand in vitro translation to determine decipher thecodons.

• E.g. UUUUUUUU -> phenylalanine only– UCUCUCUCUCU -> mix of leucine and serine

• Why is it a mixture?

The code is redundant

• Several differentcodons encodethe same aminoacid

Second mRNA baseU C A G

U

C

A

G

UUUUUCUUAUUG

CUUCUCCUACUG

AUUAUCAUAAUG

GUUGUCGUAGUG

Met orstart

Phe

Leu

Leu

lle

Val

UCUUCCUCAUCG

CCUCCCCCACCG

ACUACCACAACG

GCUGCCGCAGCG

Ser

Pro

Thr

Ala

UAU

UACUGUUGC

Tyr Cys

CAUCACCAACAG

CGUCGCCGACGG

AAUAACAAAAAG

AGU

AGCAGAAGG

GAUGACGAAGAG

GGUGGCGGAGGG

UGG

UAAUAG Stop

Stop UGA Stop

Trp

His

Gln

Asn

Lys

Asp

Arg

Ser

Arg

Gly

U

CA

GUCAG

UCAG

UCAG

Firs

t mR

NA

bas

e (5′ e

nd)

Third

mR

NA

bas

e (3′ e

nd)

The code is comma free

• No punctuation between words.– Therefore deletions cause frameshifts

• It does have start and stop signals,however.– Start: AUG– Stop: UAG, UAA, UGA

Translation: the basic conceptTRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

Polypeptide

Aminoacids

tRNA withamino acidattachedRibosome

tRNA

Anticodon

mRNA

Trp

Phe Gly

A G C

A A A

CC

G

U G G U U U G G C

Codons5′ 3′

4

The structure of transfer RNA (tRNA)3′ACCACGCUUAA

GACACCU

GC

*G U G U

CUGAG

GU

A

AA G

UC

AGACC

C G A GA G G

G

GACUCAU

UUAGGCG5′

Amino acidattachment site

Hydrogenbonds

Anticodon

*

*

**

*

**

*

**

*

tRNAAmino acidattachment site

Hydrogen bonds

Anticodon Anticodon

(b) Three-dimensional structure

A A G

5′3′

3′ 5′

Symbol used in this book

(c)

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

E P A

mRNA5′

3′

Ribosomes

Growingpolypeptide

tRNAmolecules Large

subunit

SmallsubunitRNA + protein.

We now think that RNAcatalyzes the reactions.

P site (Peptide site)

E site (Exit site)

mRNAbinding site

Largesubunit

Smallsubunit

E P A

A site (Acceptor site)

EPA model of a ribosome

5

Amino end Growing polypeptide

Next amino acidto be added topolypeptide chain

tRNA

mRNA

Codons

3′

5′

New amino acids are added to thecarboxyl end of the polypeptide

The initiation of translation

2

Initiator tRNA

mRNA

mRNA binding siteSmallribosomalsubunit

Largeribosomalsubunit

Translation initiation complex

P site

GDPGTP

Start codon

1

MetMet

U A CA U G

E A

3′

5′

5′

3′

3′5′ 3′5′

Small subunit binds justupstream of start

Special initiator methioninebinds to the start codon (AUG)

The large subunit can nowbind to make the activeribosome

ElongationAmino endof polypeptide

mRNA

tRNA withappropriateanticodon binds atA site

Ribosome ready fornext aminoacyl tRNA

Translocaton.Everything moves leftone notch. Polypeptideis now at P. EmptytRNA leaves the E site.

E

P A

E

P A

E

P A

E

P A

GDPGTP

GTP

GDP

2

2

site site5′

3′

3

2

1TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

Peptide bondformed. Growingchain is now at A

Termination

Release factor

Freepolypeptide

Stop codon(UAG, UAA, or UGA)

1 2 3

5′

3′

5′

3′

5′

3′

Stop codon hasno matchingtRNA

Release factorbinds andseparates thepolypeptidefrom the tRNA

The ribosomeseparatesfrom mRNA

6

4

An aminoacyl-tRNA synthetase joins aspecific amino acid to a tRNA

Amino acid

ATP

Adenosine

Pyrophosphate

Adenosine

Adenosine

tRNA

P P P

P

P Pi

PiPi

P

AMP

AppropriatetRNA bonds to amino

Acid, displacingAMP.

Active site binds theamino acid and ATP. 1

3

Aminoacyl-tRNAsynthetase (enzyme)

Activated amino acidis released by the enzyme.

See the Animation

• www.dnai.org

Inhibition of protein synthesisToxin Mode of action Target

Puromycinforms peptidyl-puromycin, prevents

translocationProcaryotes

Tetracyclineblocks the A-site, prevents binding of aminoacyl

tRNAsProcaryotes

Chloramphenicol blocks peptidyl transfer Procaryotes

Cycloheximide blocks peptidyl transferase Eucaryotes

Streptomycin inhibits initiation at high concentrations Procaryotes

Diphtheria toxin catalyzes ADP-ribosylation of residue in eEF2 Eucaryotes

Erythromycin binds to 50S subunit, inhibits translocation Procaryotes

Ricininactivates 60S subunit, depurinates an

adenosine in 23S rRNAEucaryotes

NOTE: Prokaryotes (this generally includes proteinsynthesis in mitochondria and chloroplasts)

Summary of transcription andtranslation in a eukaryotic cell

TRANSCRIPTION RNA is transcribedfrom a DNA template.

DNA

RNApolymerase

RNAtranscript

RNA PROCESSING In eukaryotes, theRNA transcript (pre-mRNA) is spliced andmodified to producemRNA, which movesfrom the nucleus to thecytoplasm.

Exon

Poly-A

RNA transcript(pre-mRNA)

Intron

NUCLEUSCap

FORMATION OFINITIATION COMPLEX

After leaving thenucleus, mRNA attachesto the ribosome.

CYTOPLASM

mRNA

Poly-A

Growingpolypeptide

Ribosomalsubunits

Cap

Aminoacyl-tRNAsynthetase

AminoacidtRNA

AMINO ACID ACTIVATION

Each amino acidattaches to its proper tRNAwith the help of a specificenzyme and ATP.

Activatedamino acid

TRANSLATION A succession of tRNAsadd their amino acids tothe polypeptide chainas the mRNA is movedthrough the ribosomeone codon at a time.(When completed, thepolypeptide is releasedfrom the ribosome.)

Anticodon

A CC

A A AUGGUU UA UG

U ACE A

Ribosome

1

Poly-A

5′

5′

3′

Codon

2

3 4

5