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
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