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Translation

Protein Synthesis: Ch 17From : Kevin Brown – University of Florida

+Protein Synthesis

Translation from mRNA

Codons to amino acids

http://www.youtube.com/watch?v=suN-sV0cT6c

+mRNA transcript organization

1) Open reading frame: codes for protein5’end has translational start site, 3’ end

has translational stop site

2) 5’ UTR – untranslated region (upstream of start site)

3) 3’ UTR – untranslated region (downstream of stop site)

+Typical mRNA molecule structure

+Codons

Codons are read from 5’ – 3’ in triplets

Genetic code

61 codons for amino acids, 20 amino acids

Amino acid sequence is determined by the complementary base pairing between mRNA codons and tRNA anticodons

+Translation – tRNA moleculetRNA – Transfer RNA – transfers amino

acids from cytoplasm to ribosome, where the amino acids get added onto a growing polypeptide chain.

Anticodon – triplet code found on tRNA, that is complementary to a specific mRNA codon.

Made of a single RNA strand that folds back on itself to form a 3 D structure – L-shaped.

+tRNA

64 codons

45 tRNA

Why?

Last base on tRNA anticodon is flexible – so some tRNA anticodons can recognize more than one codon

+Wobble

Non standard base pairing at 3’ end of codon – flexibilityMany tRNAs have inosine base at 5’ end of anticodon-Inosine can pair with A, U, or G

+tRNA bind to specific amino acid

Aminoacyl tRNA synthetase joins a particular amino acid to a tRNA through covalent bonding

There are 20 of these enzymes

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

PPi

i

i

Adenosine

tRNA

AdenosineP

AMP

Aminoacyl tRNA(“charged tRNA”)

+Start signalsTranslational start signal

5’ AUG 3’ , which codes for Methionine

Which AUG is the start signal?Bacteria – often have Shine-Delgarno

sequence upstream from AUG

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Eukaryotes – no Shine Delagarno, but a loose sequence – Kozak sequence

+rRNA - ribosomal RNA

Ribosome is made up of rRNA & proteins

Ribosome – the site of protein synthesis

Eukaryotes: Large subunit – 60 S, Small subunit – 40 S

Bacteria: Large subunit – 70S , Small subunit – 30 S

+Ribosome Schematic

Exit tunnel

A site (Aminoacyl-tRNA binding site)

Smallsubunit

Largesubunit

P A

P site (Peptidyl-tRNAbinding site)

mRNAbinding site

E site (Exit site)

E

+Protein Synthesis - Initiation

InitiatortRNA

mRNA

5

53Start codon

mRNA binding site

3

Translation initiation complex

5 33U

UA

A GC

P

P site

i

GTP GDP

Met Met

Largeribosomalsubunit

E A

5

Amino end

mRNAE

5 Codons

3

tRNA

Growing polypeptide

Next aminoacid to beadded topolypeptidechain

+Protein synthesis - ElongationAmino end ofpolypeptide

mRNA

5

E

Asite

3

E

GTP

GDP P i

P A

E

P A

GTP

GDP P i

P A

E

Ribosome ready fornext aminoacyl tRNA

Psite

1) Codon recognition

2) Peptide bond formation

3) Translocation

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Releasefactor

Stop codon(UAG, UAA, or UGA)

3

5

3

5

Freepolypeptide

2 GTP

5

3

2 GDP 2 iP

Protein synthesis - Termination

+Translation animation

http://www.youtube.com/watch?v=Ikq9AcBcohA

+Energy usage during Translation tRNA Charging – ATP to AMP + PPi for each

amino acid covalently bonded to tRNA

Initiation – 1 ATP to ADP + Pi and 1 GTP to GDP + Pi hydrolyzed during formation of initiation complex

Elongation – 2 GTP hydrolyzed per amino acid 1 for codon recognition 1 for energy for translocation of tRNA from A to

P

Termination – 1 GTP to GDP + Pi hydrolyzed in termination

+Completing the protein

Folding (may be helped w/chaperone protein)

Post-translational modifications:Chemical modifications – add groupsRemove amino acids from leading end

of polypeptide chainCleavage of protein

+Some polypeptides get targeted to specific locationsAll ribosomes start free

If polypeptide gives signal, ribosome binds to ER.

Signal peptide is at leading (NH3) end of polypeptide

Signal recognition particle (SRP) – recognized signal peptide and brings into receptor protein in ER

Protein either is in ER lumen, or may end up as part of membrane

+MutationsChanges in genetic information of cell

(or virus)

Small scale – few nucleotides:Substitution, insertion, deletionFrameshift are most damaging

Large scale – chromosomal changes

+Translation

http://www.dnalc.org/view/15501-Translation-RNA-to-protein-3D-animation-with-basic-narration.html