Central Dogma

Cytoplasm of eukaryoteCytoplasm of prokaryote

DNA mRNA Proteintranscription translation

replication

Translation converts sequence of bases in mRNAto sequence of amino acids in polypeptide

Lecture 12 - Translation

*Translation Overview

Genetic Code

tRNA

Charging reactions

Ribosome

Protein SynthesisInitiation - Prokaryotes vs EukaryotesElongationTermination

Overview: Players in Translation

Messenger RNA (mRNA)

RibosomeProteinsRibosomal RNA (rRNA)

Transfer RNA (tRNA)

Other molecules (proteins, GTP etc.)

CGAT -- linear sequence of 4 basesDNA

RNA CGAU -- linear sequence of 4 bases

PROTEIN KRHSTNQAVILMFYWCGPDElinear sequence of 20 amino acids

convert mRNA sequence to amino acid sequence

Genetic Code

How many bases must be read at one time in order to have a unique code for each amino acid?

codons

Triplet Code

Frameshift mutations

There are 3 possible frames to read a mRNA sequence

Universal (almost) Genetic Code

80 nucleotides

Acceptor StemAcceptor Stem

tRNA

ECB 7-23ECB 7-23

Codon - anticodon base pairing

mRNA

codon anticodon antiparallel

5’3’

Genetic code is degenerate (redundant)

Wobble in 3rd position of codon

Aminoacyl-tRNA Synthetase enzymes

One tRNA synthetase for each amino acid

Synthetase binds tRNA - specificity conferred by the anticodon loop and the acceptor stem.

How does the correct aa become attached to the

corresponding tRNA?

“charged tRNA”

Charging reaction and base pairing

Energetics - ATP to AMP; equivalent to 2 ATPs to charge tRNA

ECB 7-26ECB 7-26

Amino acid is bonded to 3’ OH of tRNA

Genetic Code

Translates linear sequence of 4 bases (RNA) to linear sequence of 20 amino acids.

Codon 3-base sequence on mRNA that specifies an amino acid

Reading Frame Grouping of nucleotide sequence into codons (3 reading frames possible, only one is used)

Terminology

Anticodon 3-base sequence on tRNA that specifies an amino acid

Charging Reaction Adds amino acid to tRNA

EukaryoticEukaryotic ribosomesribosomes

Prokaryotic ribosomesProkaryotic ribosomes

See ECB 7-28

Ribosome has 1 binding site for mRNA and 3 for tRNA

mRNA binds small subunitmRNA binds small subunit

tRNAs bind both tRNAs bind both subunitssubunits(at interface)(at interface)

ECB 7-29

Translation Overview

Genetic Code

tRNA

Charging reactions

Ribosome

*Protein SynthesisInitiation - Prokaryotes vs EukaryotesElongationTermination

Lecture 12 - Translation

Shine-Delgarno sequence is 5’ (upstream) of initiation codon (AUG) on mRNA(in 5’ UTR)

---GGAGGA------GGAGGA---mRNAmRNA -5’

Shine-Delgarno sequence

---ACCUCCUUUA------ACCUCCUUUA---rRNArRNA -3’

Initiation in Prokaryotes

mRNA binds to small ribosomal subunit by base pairing to 16S rRNA

GDP + Pi

Initiation in Prokaryotes30S

Initiation factorsInitiation factors

30S initiation30S initiationcomplexcomplex

50S

70S initiation70S initiationcomplexcomplex

30S

fmet tRNAGTPIF2

InitiationInitiation codoncodonS-DS-D

AUG determines reading frame

Translation can be initiated at several sites on prokaryotic mRNA

Prokaryotes - In polycistronic mRNA coded by an operon, eachcoding region must have Shine-Delgarno sequence and AUG

ECB7-29

ECB 7-33

Initiation in eukaryotes

ECB 7-32

Stepwise addition of amino acids

Elongation factors (EFs) are required

3 Key steps: 1. Entry of aminoacyl-tRNA

2. Formation of a peptide bond

3. Translocation - movement of ribosome with respect to the mRNA

3 tRNA binding sites: A, P, E

A site = Aminoacyl site, accepts new tRNA

P site = Peptidyl site, tRNA with growing polypeptide chain

E site = Exit site, release of uncharged tRNA

Translation Elongation (eukaryotic and prokaryotic)

Start with tRNA + peptide chain in P site (only a singe aa if chain just initiated)

E P A

E P A

Three steps in Three steps in elongationelongation

ECB 7-31

N- to C-terminus synthesis

Peptidyltranserase reaction- Peptide Bond Formation

Proks and euks

Does not require input of energy

TerminationTermination

3 stop codons; UAG, UGA, UAA3 stop codons; UAG, UGA, UAAECB 7-34

Protein synthesis is energetically expensive…

• Charging aa-tRNA: 2 ATP (ATP -> AMP+2Pi)…

• Binding of aa-tRNA/proofreading: 1 GTP…

• Translocation of ribosome 1 codon towards 3’ end of mRNA: 1 GTP…

• Total of at least 4 high energy bonds/aa added…

• As much as 80% of cells energy devoted to protein synthesis!

Peptidyl-tRNA in P site…

A site is empty…

Adapted from ECB figure 7-31

Polypeptide elongation

Polypeptide elongation

Step 1: Complex of aa-tRNA andEF1-GTP binds in A-site…

Polypeptide elongation

Polypeptide elongation

Polypeptide elongation

Requirement for GTP hydrolysis and release of EF1 before peptide bond formation imposes a time delay…allowing wrong aa-tRNAs to dissociate from ribosome = proofreadingproofreading

Polypeptide elongation

Step 3a: Large subunit shifts relative to small subunit and mRNA…

Step 2: Peptide bond formed (energy of 2 ATP from charging of aa-tRNA).

Polypeptide elongation

Step 3b: Small subunit moves 1 codon (3 nucl.) towards 3’ end. Empty tRNA is ejected.

GTP GDP + PGTP GDP + Pii

Polypeptide elongation

Prokaryotes: ~20 aa/sec…

Eukaryotes: ~ 2 aa/sec…

Polypeptide elongation

07.6-translation_II.mov

Polyribosomes

Multiple ribosomes translating one mRNA

5’ to 3’

ECB 7-35

Antibiotics that block prokaryotic protein synthesis