Central Dogma Cytoplasm of eukaryote Cytoplasm of prokaryote DNAmRNA Protein transcription...

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