24-1 Chapter 13 From Gene to Protein. 24-2 Beadle & Tatums Experiment Used Neurospora (mold) to...

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Chapter 13From Gene to Protein

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Beadle & Tatum’s Experiment

• Used Neurospora (mold) to determine effect mutation• They isolated mutants that required addition of arginine to

survive

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Beadle & Tatum’s Experiment:Results

The wild-type strain required only the minimal medium for growth. The three classes of mutants had different growth requirements

Class IMutants

Class IIMutants

Class IIIMutantsWild type

Minimal medium(MM)(control)

MM +Ornithine

MM +Citrulline

MM +Arginine(control)

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CONCLuSION Because each of their mutants was mutated in a single gene, Beadle & Tatum concluded that each mutated gene must normally dictate the production of one enzyme. (Notice that a mutant can grow only if supplied with a compound made after the defective step.) Class I

Mutants(mutationin gene A)

Class IIMutants(mutationin gene B)

Class IIIMutants(mutationin gene C)Wild type

Gene A

Gene B

Gene C

Precursor Precursor PrecursorPrecurso

r

Ornithine Ornithine Ornithine Ornithine

Citrulline Citrulline Citrulline Citrulline

Arginine Arginine Arginine Arginine

EnzymeA

EnzymeB

EnzymeC

A A A

B B B

C C C

Beadle & Tatum’s Experiment:Conclusions

• One gene one enzyme hypothesis• Replaced by one gene one protein hypothesis• That too has been replaced…

Genes can code for a polypeptide or an RNA sequence

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RNA Structure & Function

• RNA – Composed of nucleotides– Single-strand– found in both nucleus &

cytoplasm

• Structure of RNA– Phosphate– 5-C sugar--Ribose– 4 possible nitrogen bases

• Adenine and guanine• Cytosine and uracil (uracil

replaces thymine)

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Transcription

• Synthesis of RNA from a DNA template

• Types of RNA– mRNA- carries genetic

information from the DNA out to ribosomes

– rRNA- composes ribosomes, site of protein assembly

– tRNA- brings in amino acids to the ribosomes

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Transcription: initiation, elongation, and termination

PromoterTranscription unit

RNA polymerase

Start point

53

35

DNA

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RNA polymerase

Start point

53

35

35

53

Unwound

DNA

RNA

transcript

Template strand of DNA

1 Initiation. After RNA polymerase binds to

the promoter, the DNA strands unwind, and

the polymerase initiates RNA synthesis at the

start point on the template strand.

DNA

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RNA polymerase

Start point

53

35

35

53

53

35

5

Rewound

RNA

RNA

transcript

3

Unwound

DNA

RNA

transcript






2 Elongation. The polymerase moves downstream, unwinding the

DNA and elongating the RNA transcript 5 3 . In the wake of

transcription, the DNA strands re-form a double helix.

DNA

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RNA polymerase

Start point

53

35

35

53

53

35

53

35

5

5

Rewound

RNA

RNA

transcript

3

3

Completed RNA transcript

Unwound

DNA

RNA

transcript






3 Termination. Eventually, the RNA

transcript is released, and the

polymerase detaches from the DNA.

2 Elongation. The polymerase moves downstream, unwinding the

DNA and elongating the RNA transcript 5 3 . In the wake of

transcription, the DNA strands re-form a double helix.

DNA

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Elongation

RNApolymerase

Non-templatestrand of DNA

RNA nucleotides

3 end

C A E G C AA

U

T A G G T TA

AC

G

U

AT

CA

T C C A AT

T

GG

3

5

5

Newly madeRNA

Direction of transcription(“downstream) Template

strand of DNA

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Special Case:Transcription in Eukaryotes

• Transcription Factors are necessary to bind RNA Polymerase to promoter

• Transcription initiation complex

• “TATA box”– Part of

promoter sequence

• DNAi animation

Prokaryotes

• Start Translation while transcription is still going on

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RNA Processing

• Primary mRNA – Contains bases

complementary to both intron and exon segments of DNA

• “Capping” mRNA (after transcrption)– 5’ cap (modified GTP)

• Prevents degradation by enzymes

• “Attach here” sign– 3’ (polyA tail)

• Inhibits degradation• Spliceosome

– RNA splicing removes intron sequences

– Ribozyme (RNA enzyme) splices exon segments together

– Animations

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html

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Exons, Protein Domains and Quaternary Structure

GeneDNA

Exon 1 Intron Exon 2 Intron Exon 3

Transcription

RNA processing

Translation

Domain 3

Domain 1

Domain 2

Polypeptide

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“Dictionary” of the Genetic Code

• Triplet code- codon– 3 sequential Nitrogen bases in mRNA

• Redundancy (AKA: “wobble”)– (some AA have different codons)– Protection against mutations

• 64 different mRNA codons– 61 code for different amino acids – Stop codons (UAA, UAG, UGA) signal

termination of translation– Start codon methionine (starts translation)

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Messenger RNA codonsSecond mRNA base

U C A G

U

C

A

G

UUU

UUC

UUA

UUG

CUU

CUC

CUA

CUG

AUU

AUC

AUA

AUG

GUU

GUC

GUA

GUG

Met orstart

Phe

Leu

Leu

lle

Val

UCU

UCC

UCA

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

AGC

AGA

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

C

A

G

U

C

A

G

U

C

A

G

U

C

A

G

Fir

st m

RN

A b

ase

(5

end

)

Th

ird

mR

NA

bas

e (3

en

d)

Glu

Evolution of the Genetic Code

• The genetic code is nearly universal– Shared by organisms from the simplest bacteria to the most

complex animals

• In laboratory experiments– Genes can be transcribed and translated after being

transplanted from one species to another

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Another format:The “wheel”

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Aminoacyl-tRNA synthetase:Joining AA and tRNA

Amino acid

ATP

Adenosine

Pyrophosphate

Adenosine

Adenosine

Phosphates

tRNA

P P P

P

P Pi

Pi

Pi

P

AMP

Aminoacyl tRNA(an “activatedamino acid”)

AppropriatetRNA covalentlyBonds to aminoAcid, displacing

AMP.

Active site binds theamino acid and ATP. 1

3

Activated amino acidis released by the enzyme.

4

Aminoacyl-tRNAsynthetase (enzyme)

ATP loses two P groupsand joins amino acid as AMP.2

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Translation: Overview

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

Polypeptide

Aminoacids

tRNA withamino acidattached

Ribosome

tRNA

Anticodon

mRNA

Trp

Phe Gly

AG C

A A A

CC

G

U G G U U U G G C

Codons5 3

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Ribosome

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

PolypeptideExit tunnel

Growingpolypeptide

tRNAmolecules

EP A

Largesubunit

Smallsubunit

mRNA

Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins.

(a)

53

Ribosome Association and Initiation of Translation

– Brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome

Largeribosomalsubunit

The arrival of a large ribosomal subunit completes the initiation complex. Proteins called initiationfactors (not shown) are required to bring all the translation components together. GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available to the tRNA bearing the next amino acid.

2

Initiator tRNA

mRNA

mRNA binding site Smallribosomalsubunit

Translation initiation complex

P site

GDPGTP

Start codon

A small ribosomal subunit binds to a molecule of mRNA. In a prokaryotic cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. An initiator tRNA, with the anticodon UAC, base-pairs with the start codon, AUG. This tRNA carries the amino acid methionine (Met).

1

MetMet

U A CA U G

E A

3

5

53

35 35

Figure 17.17

Elongation– Amino acids are added one by one to the

preceding amino acid

Figure 17.18

Amino endof polypeptide

mRNA

Ribosome ready fornext aminoacyl tRNA

E

P A

E

P A

E

P A

E

P A

GDPGTP

GTP

GDP

2

2

sitesite5

3

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

Codon recognition. The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysisof GTP increases the accuracy andefficiency of this step.

1

Peptide bond formation. An rRNA molecule of the large subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the tRNA in the A site.

2

Translocation. The ribosome translocates the tRNA in the A site to the P site. The empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs,bringing the next codon to be translated into the A site.

3

Termination of Translation• The final stage of translation is termination

– When the ribosome reaches a stop codon in the mRNA

Figure 17.19

Release factor

Freepolypeptide

Stop codon(UAG, UAA, or UGA)

5

3 3

5

35

When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA.

1 The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome.

2 3The two ribosomal subunits and the other components of the assembly dissociate.

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DNAi

• Watch Translation Animation

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Polyribosomes

Growingpolypeptides

Completedpolypeptide

Incomingribosomalsubunits

Start of mRNA(5 end)

End of mRNA(3 end)

Polyribosome

An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes.

(a)

Ribosomes

mRNA

This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

0.1 µm(b)

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Signal mechanism:Targeting proteins to the ER

The rest ofthe completedpolypeptide leaves the ribosome andfolds into its finalconformation.

Polypeptidesynthesis beginson a freeribosome inthe cytosol.

An SRP binds to the signal peptide, halting synthesismomentarily.

The SRP binds to areceptor protein in the ERmembrane. This receptoris part of a protein complex(a translocation complex)that has a membrane poreand a signal-cleaving enzyme.

The SRP leaves, andthe polypeptide resumesgrowing, meanwhiletranslocating across themembrane. (The signalpeptide stays attachedto the membrane.)

The signal-cleaving enzymecuts off thesignal peptide.

1 2 3 4 5 6

Ribosome

mRNASignalpeptide

Signal-recognitionparticle(SRP) SRP

receptorprotein

Translocationcomplex

CYTOSOL

Signalpeptideremoved

ERmembrane

Protein

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Gene Mutations

• Permanent changes in DNA

• Point mutations– Change in a single base

pairIn the DNA, themutant templatestrand has an A where the wild-type template has a T.The mutant mRNA has a U instead of an A in one codon.

The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu).

Mutant hemoglobin DNAWild-type hemoglobin DNA

mRNA mRNA

Normal hemoglobinSickle-cell

hemoglobinGlu Val

C T T C A T

G A A G U A

3 5 3 5

5 35 3

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Substitutions

Wild type

A U G A A G U U U G G C U A AmRNA 5Protein Met Lys Phe Gly Stop

Carboxyl endAmino end

3

A U G A A G U U U G G U U A A

Met Lys Phe Gly

Base-pair substitutionNo effect on amino acid sequence

U instead of C

Stop

A U G A A G U U U A G U U A A

Met Lys Phe Ser Stop

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

Met Stop

Missense A instead of G

NonsenseU instead of A

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Base-pair insertion or deletion

mRNA

Protein

Wild type

A U G A A G U U U G G C U A A5

Met Lys Phe Gly

Amino end Carboxyl end

Stop

Base-pair insertion or deletion

Frameshift causing immediate nonsense

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

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

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

Met Stop

U

Met Lys Leu Ala

Met Phe GlyStop

MissingA A G

Missing

Extra U

Frameshift causing extensive missense

Insertion or deletion of 3 nucleotides:no frameshift but extra or missing amino acid

3

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Frameshift mutation

• Change “coding frame” (how the codon is read)

• Silent mutations– No change in Amino

Acid (redundancy)• Nonsense mutations

– Changes whole amino acid sequence protein can’t function

• Missense mutations– Change in only a few

amino acids protein can function, but causes problems (sickle cell anemia)

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Mutagens:Physical, Chemical, Environmental

• Tobacco (lung, throat, mouth, larynx, esophagus)

• UV (skin)• Alcohol ( mouth, throat,

esophagus, larynx & liver)• Radiation (depends on

area being irradiated)• Diet• Occupational Hazards

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Transposons

• Discovered by Barbara McClintock• “Jumping Genes” genes that can “move” within or between

chromosomes– Require Transposase—which they make themselves!

• May increase or decrease gene expression • May destroy gene expression

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