Ch 17 Gene Expression II: Translation mRNA->Protein

LE 17-4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNA strand(template)

3

TRANSCRIPTION

Codon

mRNA

TRANSLATION

Protein

Amino acid

35

5DNA

RNA

Protein

Review

LE 17-4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNA strand(template)

3

TRANSCRIPTION

Codon

mRNA

TRANSLATION

Protein

Amino acid

35

5DNA

RNA

Protein

Review

Cracking the Code

• 64 codons– decoded by the mid-1960s

•Genetic code–redundant but not ambiguous; no codon specifies more than one amino acid (but one amino acid may have >1 codon)

•Codons– must be read in the correct reading frame in order for the specified polypeptide to be produced

LE 17-5Second mRNA base

Fir

st

mR

NA

ba

se

(5 e

nd

)

Th

ird

mR

NA

ba

se

(3 e

nd

)

Genetic Code Codon Table

Find an example of redundancy in the genetic code.

Which amino acid does not have redundant codons?

Is there a pattern to redundant codons?

Evolution of the Genetic Code

• Genetic code– nearly universal: shared by the simplest bacteria,

plants, fungi and animals

• Genes can be transcribed and translated after being transferred from one species to another

Mechanism of Translation

• Ribosomes- Bind messenger (mRNA)

- Attract transfer RNA (tRNA) to mRNA

- tRNA covalently linked to specific amino acid (aa-tRNA)

-Complementary basepairs form between mRNA and aa-tRNA (codon-anticodon interactions)

-Enzyme in ribosome catalyzes peptide bond between amino acids- -> polypeptide chain grows

LE 17-14a

Amino acidattachment site

Hydrogenbonds

3

5

Two-dimensional structureAnticodon

Amino acidattachment site

35

Hydrogenbonds

Anticodon Anticodon

Symbol used in this bookThree-dimensional structure

3 5

tRNA structure

~ 80 nt long

Three different schematics

In what ways do theyconvey the sameand different information?

LE 17-13

Polypeptide

tRNA withamino acidattached

Ribosome

tRNA

Anticodon

35

mRNA

Aminoacids

Codons

1. a correct match between tRNA and an amino acid- Catalyzed by aminoacyl-tRNA synthetase

Accurate translation requires two steps

2. a correct match between the tRNA anticodon and an mRNA codon

LE 17-15Amino acid Aminoacyl-tRNA

synthetase (enzyme)

Pyrophosphate

Phosphates

tRNA

AMP

Aminoacyl tRNA(an “activatedamino acid”)

1.

Ribosomes

• Facilitate specific coupling of anticodons with codons

• Ribosomal structure

– Two ribosomal subunits (large and small)

• Made of proteins (ribosomal proteins) and ribosomal RNA (rRNA)

Form binding sites for mRNA and aa-tRNA

Draw

LE 17-16a

tRNAmolecules

Exit tunnelGrowingpolypeptide

Largesubunit

mRNA 3

Computer model of functioning ribosome

Smallsubunit

5

E P A

LE 17-16b

P site (Peptidyl-tRNAbinding site)

E site (Exit site)

mRNAbinding site

A site (Aminoacyl-tRNA binding site)

Largesubunit

Smallsubunit

Schematic model showing binding sites on ribosome

E P A

LE 17-16c

Amino end

mRNA

5

3

Growing polypeptide

Next amino acidto be added topolypeptide chain

tRNA

Codons

Schematic model with mRNA and tRNA

E

Ribosome translates 5’ to 3’ on mRNA.

Polypeptide chain grows amino end first, carboxyl end last.

Building a Polypeptide

• The three stages of translation:– Initiation– Elongation– Termination

•All three stages require protein translation factors

Ribosome Association and Initiation of Translation

1. Small ribosomal subunit binds mRNA and special initiator tRNA (met-tRNAi)

(carries the amino acid methionine)

2. Small subunit scans along the mRNA until first start codon (AUG).

3. Initiation factors bring in large subunit

initiator tRNA occupies the P site.

LE 17-5Second mRNA base

Fir

st

mR

NA

ba

se

(5 e

nd

)

Th

ird

mR

NA

ba

se

(3 e

nd

)

Genetic Code Codon Table

MemorizeStartCodon

LE 17-17

Met

GTPInitiator tRNA

mRNA

53

mRNA binding site

Smallribosomalsubunit

Start codon

P site

5 3

Translation initiation complex

E A

Largeribosomalsubunit

GDP

Met

Elongation of the Polypeptide Chain

- Amino acids are added one by one to the preceding amino acid

-Elongation factors facilitate

- codon recognition

- peptide bond formation

- translocation

LE 17-18

Ribosome ready fornext aminoacyl tRNA

mRNA

5

Amino endof polypeptide

E

Psite

Asite

3

2

2 GDP

E

P A

GTP

GTP

GDP

E

P A

E

P A

1. Recognition

2. Peptide bondformation

3. Translocation

Termination of Translation

- Occurs when stop codon in mRNA reaches A site of ribosome

- A site accepts protein called release factor

-Release factor causes addition of water molecule instead of amino acid

- Polypeptide released, ribosomal subunits dissociate and fall off mRNA

LE 17-5Second mRNA base

Fir

st

mR

NA

ba

se

(5 e

nd

)

Th

ird

mR

NA

ba

se

(3 e

nd

)

Genetic Code Codon Table

MemorizeStopCodons

LE 17-19

3

The release factor hydrolyzes thebond between the tRNA in theP site and the last amino acid of thepolypeptide chain. The polypeptideis thus freed from the ribosome.

The two ribosomal subunitsand the other componentsof the assembly dissociate.

Releasefactor

Stop codon(UAG, UAA, or UGA)

5

3

5

3

5

Freepolypeptide

When a ribosome reaches a stopcodon on mRNA, the A site of theribosome accepts a protein calleda release factor instead of tRNA.

Let’s translate a mRNA…5’ cgaggucaaugcccuauguuuagccc 3’

Bracket each codonin the open readingframe (ORF).

What is theanticodon forthe secondcodon in the ORF?

Write the aminoacid below each codon.

5’3’

I’m complicated but onceyou get to know meI’m really pretty nice.Any questions?

Can a transcript (mRNA) be translated by multipleribosomes simultaneously?

Polyribosomes

• -a single mRNA (transcript) is translated by many ribosomes simultaneously

• mRNA+ bound ribosomes= polyribosomes or polysome

• Allows fast synthesis of many copies a polypeptide

LE 17-20

Ribosomes

mRNA

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

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

Incomingribosomalsubunits

Growingpolypeptides

End ofmRNA(3 end)

Start ofmRNA(5 end)

Polyribosome

CompletedpolypeptidesPolyribosome

or Polysome

Consider: When a eukaryotic message is transcribed, processed and transported to the cytosol, is it immediately translatedinto protein?

When would a cell need a polypeptide immediately?When would a cell want to delay translation? Examples?

What strategy could one use to determine whether a mRNA was being actively translated?

Hint: consider mass

Subject cell homogenate to differential centrifugation-Heavy polysomes will pellet

-Light untranslated mRNA in supernatant

Polysomes in Prokaryotes

Where and when are transcripts translated in prokaryotes?

Coupled transcription and translation

LE 17-22

RNA polymerase

DNA

Polyribosome

RNApolymerase

Direction oftranscription

mRNA

0.25 m

DNA

Polyribosome

Polypeptide(amino end)

Ribosome

mRNA (5 end)

Targeting Polypeptides to Specific Locations

In eukaryotes, what are the two populations of ribosomes?

Free, soluble in cytosol synthesize soluble proteins

Bound to rER- synthesize secreted or membrane bound proteins

- tagged with signal peptide at amino end

LE 17-21

Ribosomes

mRNASignalpeptide

Signal-recognitionparticle(SRP)

SRPreceptorprotein

CYTOSOL

ER LUMEN Translocationcomplex

Signalpeptideremoved

ERmembrane

Protein

Signal peptide targets polypeptides to ERfinal polypeptide destined for secretion or membrane

Is the molecular weight of a secreted protein different than the predicted translation product of its mRNA?

Effect of mutations on gene expression

Any change in the genetic material of a cell or virus

What is a mutation?

Types of mutations

Point: a single nucleotide change

-substitution gcca->gcga

-deletion gcca->gca

-insertion gcca->gacca

Also, breaks, translocations, inversions as reviewed previously

LE 17-23

Wild-type hemoglobin DNA

3 5 53

Mutant hemoglobin DNA

Normal hemoglobin Sickle-cell hemoglobin

What kind of mutation? substitution

mRNA

5 3 35

mRNA

Transcribe into mRNA

Translate into protein

LE 17-5Second mRNA base

Fir

st

mR

NA

ba

se

(5 e

nd

)

Th

ird

mR

NA

ba

se

(3 e

nd

)

Genetic Code Codon Table

MemorizeStartCodon

Substitutions

• Missense mutations– Change codon to encode a different amino acid

• Nonsense mutations– Change codon to encode a stop codon

nearly always leading to a nonfunctional protein

Missense mutations are more common.Why?

LE 17-24

Base-pair substitution

No effect on amino acid sequenceU instead of C

MissenseA instead of G

NonsenseU instead of A

Stop

Amino end

Protein

5 3

Carboxyl end

Stop

Stop

Stop

mRNA

Wild type

Neutral

Change in amino acid

Premature termination

Substitutions

Insertions and Deletions

• Alters reading frame ->frameshift mutation

• Often more devastating than substitutions

LE 17-25

Base-pair insertion or deletion

Addition frameshift

Extra U

MissingDeletion frameshift

Insertion or deletion of 3 nucleotides

Missing

Stop

Stop

Amino end Carboxyl end

Stop

Wild type

mRNA

Protein5 3

Source of Mutations

• From spontaneous mutations: occur during DNA replication, recombination, or repair

• From mutagens are physical or chemical agents that can cause mutations

What is a gene? revisiting the question

• A gene is a region of DNA whose final product is either a polypeptide or an RNA molecule

LE 17-26

TRANSCRIPTION

RNA PROCESSING

RNAtranscript

5

Exon

NUCLEUS

FORMATION OFINITIATION COMPLEX

CYTOPLASM

3

DNA

RNApolymerase

RNA transcript(pre-mRNA)

Intron

Aminoacyl-tRNAsynthetase

Aminoacid

tRNA

AMINO ACID ACTIVATION

3

mRNA

A

P

E Ribosomalsubunits

5

Growingpolypeptide

E A

Activatedamino acid

Anticodon

TRANSLATION

Codon

Ribosome