Translation: RNA-Directed Polypeptide Synthesis

Translation: Like making a Honda one part at a time Translation: Machinery and Process are Complex
Protein Synthesis Animation: 3-D model (Nierhaus, 2006)

Protein Synthesis Animation

CHAPTER 30: PROTEIN SYNTHESIS (Translation) Translation: mRNA protein

LECTURE TOPICS Complexity, stages, rate, accuracy Amino acid activation [tRNA charging] tRNAs and translating the Genetic Code - Amino acid activation [tRNA charging] - codon-anticodon recognition - Wobble base pairing and code degeneracy Ribosome Structure TRANSLATION PROCESS - Prokaryotic - Eukaryotic - Inhibitors [specific steps in process]

Process Rates for Flow of Genetic Information (prokaryotic)

PROCESS DNA REPLICATION "MACHINERY" DNA Polymerase I DNA Polymerase III RATES 10 bases/sec 1000 bases/sec

TRANSCRIPTION

RNA Polymerase

50 bases/sec
synthesis of mRNA for 200 a.a. protein takes (200 x 3)/50 = 12 sec

TRANSLATION

Ribosomes

20 a.a.'s/sec
synthesis of a 200 a.a. protein takes 200/20 =10 sec

Complexity of Translation: Cast of Characters [amounts in one E.coli cell]

Translation machinery is 35% of dry weight of cell! 20,000 ribosomes 200,000 tRNAs 100,000 proteins and cofactors

Complexity of Translation: Stages and Cast of Characters

How does a ribosome work?

NH2

[Large subunit]

tRNAs

[small subunit]

tRNA Common Features

* * * *

* Modified
bases can prevent charging with a wrong amino acid

Major tRNA Structure Features

*

Compact L- Shaped Mostly base-paired A-helix Unusual base pairs (3 bases)

Bases face out for pairing

 tRNA is a compact A-helix structure! Only a few bases are NOT paired!

*tRNA Movie*

Protein (polypeptide) Synthesis is NH2

COOH

Amino terminus

1 2 2 1 1 2 3 3

COOH terminus

2 Classes of aminoacyl-tRNA Synthetases: [Recognize 2 different conformations of CCA]

How can an aminoacyl-tRNA synthetase discriminate between similar amino acids??

Ex: Threonine tRNAThr amino acid charging (Activation) [Discriminate between Thr, Val, Ser]

Threonyl-tRNAThr synthetase

(CCA)

tRNAThr

1st step is formation of a.a.-AMP-enzyme Intermediate: [activation is an ATP requiring reaction with PPi as a product]

Threonyl tRNAThr synthetase Proofreading (Error correcting)

Mistakes: Actually leaves Serine (1/100 a.as)
Hydrolyzes wrong amino acid (Ser)

a.a-AMP-Enzyme

tRNA - mRNA recognition determined by?

[YES] codon-anticodon base-pairing [NO] what amino acid is on the tRNA

If Ala-tRNACys used: get alanine where cysteine should be

CHAPTER 30: PROTEIN SYNTHESIS (Translation) Translation: mRNA protein

LECTURE TOPICS tRNAs and translating the Genetic Code - Wobble base pairing and code degeneracy Ribosome Structure TRANSLATION PROCESS - Prokaryotic - Eukaryotic - Inhibitors [specific steps in process]

Codon-anticodon recognition and wobble base pairing

3 5

UH2

UH2

Sometimes inosine is at 5`-end of anticodon

5
I G C

mRNA codon

3- C C

-5

Wobble I:C base pair

Wobble Hypothesis: * Dont need 61 tRNAs! * The anticodon of some tRNAs can read 2 or 3 codons

(5`)

ONLY ONLY

(3`)
Wobble basepairing

C I

Inosine wobble Base pairs

Wobble codon base (3) pairs with (5`) - I of anticodon

[3 Ala codons read by only one tRNA anticodon]

The E. coli ribosome

Small subunit

Large subunit

Subunits associated for protein synthesis

16S rRNA Structure (1542 bases long)

5` end secondary structure 3` end

3-D X- ray structure

Ribosome Structure

30S

50S

70S

30S Facing Solvent (outer surface)

More protein-rich than other side [next slide]

30S Facing 50S

[*30S Movie*]

Mostly RNA at this surface

50S ribosome subunit

Facing 30S

Transcription and translation are coupled in E.coli They happen simultaneously!

Translation process is 100% Processive:

1. Make peptide bonds 2. Relative movements (mRNA + ribosome) 3. Supply of reagents,etc.

Elongation

Termination 30S Initiation complex

Ribosome has two essential functions in protein synthesis

1 1

2 2 5

tRNA-mRNA-rRNA base-pairing interactions determine accuracy of protein synthesis.

50S ribosome subunit

30S ribosome subunit

Three tRNAs

mRNA

50S Ribosome subunit: Protein Exit Channel

Hydrophobic channel is 100 Angstrom diameter

Protein exit channel in 50S subunit: Growing protein passes through channel.

exit

exit

50S cut in half and opened like a book

Protein doesnt fold until its out of exit channel.

[E,P,A] three tRNA binding sites on 30S subunit

NH2

[Large subunit]
3 tRNAs

[small subunit]

Three tRNA binding sites on 30S subunit

CCA ends

Exit Peptidyl

Amino-acyl

Codon-Anticodon base pairing

Protein Synthesis Cycle: tRNA movements

2
E P A E P A

1 3 Initiation complex formation

E P A E P A

Protein Synthesis Animation: 3-D model (Nierhaus, 2006) Protein Synthesis Animation

rRNA-mRNA(codon)-tRNA(anticodon) base-pairing:

another accuracy factor

Purine-rich

Start

mRNAs

Initiation: Prokaryotic mRNA-16S rRNA base-pairing

Purine-rich

Start

Prokaryotic initiator fMet-tRNAf

Prokaryotic initiation complex

[IF2 for AUG start only]

Purine-rich sequence [base-pairing]

Elongation

Protein Translation Factors

EF-Tu

23S rRNA ribozyme

E P A

E P A

GTP GDP + Pi

EF-G

Termination by Release Factors (Stop codon recognition)

E P A

E P A

Elongation Factor EF-Tu: Delivers aa-tRNA to ribosome

* (GTP)

Proofreading events can occur on the ribosome before and after EF-Tu catalyzed GTPase

Correct

(On the ribosome A-site)

Elongation : Peptide Bond Synthesis

[peptide bond formed and growing peptide moves from P-site to A-site]

..

Initiator tRNA

Catalyzed by 23S rRNA ribozyme

50S Peptidyl transferase : A ribozyme activity

A site (Green) P site(red)

P site

Adenine 2451

A site

23S RNA-catalyzed peptide bond formation

A site

Translation Process: Elongation [Fixed locations of E-P-A]

EF-Tu
23S rRNA ribozyme

E P A

E P A

GTP GDP + Pi

EF-G

A] represent location of tRNAs

E P A E P A

*P [E

Translation Process: Elongation [Fixed locations of E-P-A]

EF-Tu
23S rRNA ribozyme

E P A

E P A

GTP GDP + Pi

EF-G

A] represent location of tRNAs

E P A E P A

*P [E

Translocation : EF-G
is an EF-Tu-tRNA mimic is a G-protein (as is EF-Tu)

E P A

E P

E P A E P A

New A

New A

Next tRNA to A-site

EF-G-GDP off

EF-G structure mimics the EF-Tu tRNA complex

Termination:
Protein release factor(s) recognizes a stop codon. Stimulates release of new protein

Termination: tRNA Release Factors

Eukaryotic
[no prokaryotic structures yet] is a tRNA mimic Gly-Gly-Gln anticodon may enter peptidyl transferase center and be involved in cleavage of peptidyl-tRNA bond.

Ribosome Release Factor (RRF) Also a tRNA mimic

Works with EF-G (and GTP hydrolysis) Facilitates separation of tRNA, mRNA, and ribosome subunits.

RRF

CHAPTER 30: PROTEIN SYNTHESIS (Translation) Translation: mRNA protein

LECTURE TOPICS Ribosome Structure TRANSLATION PROCESS [Short review] - Prokaryotic - Eukaryotic - Inhibitors [specific steps in process]

Eukaryotic cytoplasmic ribosome (80S)

Eukaryotic Initiation complex

EIF-2(GTP) for start AUG only

1) Ribosome binds to cap

Ignore not fMet comments

2) Moves to 1st AUG EIF2-GDP + Pi

3) Large + small subunits associate

Elongation

Eukaryotic mRNA: protein-RNA and proteinprotein interactions circularize mRNA

eIF4E binds to cap eIF4E binds to eIF4G

eIF4G binds to PABI

Antibiotic inhibitors of protein synthesis

* Common drugs for bacterial infections

Streptomycin: Inhibits initiation and causes misreading in prokaryotic ribosomes

Cyclohehimide (a common fungicide): Inhibits eukaryotic peptidyl transferase

Streptomycin Binds to 30S ribosomal subunit

Inhibits initiation Causes misreading of 5` base of codon

Ex: 5`- UUU 3` reads as: 5`- AUU 3`

Phe

Ile

Puromycin: Terminates protein synthesis An a.a-tRNA analog that binds to A-site.

Forms peptide bond with growing protein chain and is released from ribosome A-site

Ricinus communis (castor bean) is the source of ricin, a deadly poison that stops eukaryotic protein synthesis (500g is lethal human dose).
A and B chains disulfide linked B chain binds to cell and induces uptake by endocytosis A chain released in cytoplasm A chain is a Nglycosidase that removes A4324 in 28S rRNA and prevents elongation factor binding

Summary
1

4 5