Download to linear sequence of 20 amino acids.

Central Dogma
DNA
transcription
translation
mRNA
Protein
Cytoplasm of eukaryote
Cytoplasm of prokaryote
replication
Translation converts sequence of bases in mRNA
to sequence of amino acids in polypeptide
Lecture 12 - Translation
*Translation Overview
Genetic Code
tRNA
Charging reactions
Ribosome
Protein Synthesis
Initiation - Prokaryotes vs Eukaryotes
Elongation
Termination
Overview: Players in Translation
Messenger RNA (mRNA)
Ribosome
Proteins
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Other molecules (proteins, GTP etc.)
Genetic Code
convert mRNA sequence to amino acid sequence
How many bases must be read at one time in order to have a unique
code for each amino acid?
DNA
CGAT -- linear sequence of 4 bases
RNA
CGAU -- linear sequence of 4 bases
PROTEIN
KRHSTNQAVILMFYWCGPDE
linear sequence of 20 amino acids
Triplet Code
codons
There are 3 possible frames
to read a mRNA sequence
Frameshift mutations
Universal (almost) Genetic Code
Acceptor Stem
tRNA
80 nucleotides
ECB 7-23
Codon - anticodon base pairing
3’
5’
mRNA
codon
anticodon
antiparallel
Genetic code is degenerate (redundant)
Wobble in 3rd position of codon
How does the correct aa become attached to the
corresponding tRNA?
Aminoacyl-tRNA Synthetase enzymes
One tRNA synthetase for each amino acid
Synthetase binds tRNA - specificity conferred by the
anticodon loop and the acceptor stem.
Charging reaction and base pairing
“charged tRNA”
ECB 7-26
Energetics - ATP to AMP; equivalent to 2 ATPs to charge tRNA
Amino acid is bonded to 3’ OH of tRNA
Terminology
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
Anticodon
3-base sequence on tRNA that specifies an
amino acid
Charging Reaction Adds amino acid to tRNA
Reading Frame
Grouping of nucleotide sequence into codons
(3 reading frames possible, only one is used)
Prokaryotic ribosomes
Eukaryotic ribosomes
See ECB 7-28
Ribosome has 1 binding site for mRNA
and 3 for tRNA
tRNAs bind both
subunits
(at interface)
mRNA binds small subunit
ECB 7-29
Lecture 12 - Translation
Translation Overview
Genetic Code
tRNA
Charging reactions
Ribosome
*Protein Synthesis
Initiation - Prokaryotes vs Eukaryotes
Elongation
Termination
Initiation in Prokaryotes
mRNA binds to small ribosomal subunit by base pairing to 16S rRNA
rRNA
mRNA
---ACCUCCUUUA--- -3’
-5’
---GGAGGA--Shine-Delgarno sequence
Shine-Delgarno sequence is 5’ (upstream)
of initiation codon (AUG) on mRNA
(in 5’ UTR)
Initiation factors
30S
S-D
Initiation codon
Initiation in
Prokaryotes
AUG determines reading frame
50S
fmet tRNA
GTP
IF2
30S initiation
complex
70S initiation
complex
30S
GDP + Pi
Translation can be initiated at several sites
on prokaryotic mRNA
Prokaryotes - In polycistronic mRNA coded by an operon, each
coding region must have Shine-Delgarno sequence and AUG
ECB7-29
ECB 7-33
Initiation in
eukaryotes
ECB 7-32
Translation Elongation (eukaryotic and prokaryotic)
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
Three steps in elongation
E PA
E PA
Start with tRNA + peptide chain in P site
(only a singe aa if chain just initiated)
ECB 7-31
Peptidyltranserase reaction- Peptide Bond Formation
Does not require input of energy
Proks and euks
N- to C-terminus synthesis
Termination
ECB 7-34
3 stop codons; UAG, UGA, UAA
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!
Polypeptide elongation
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 and
EF1-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 aatRNAs to dissociate from ribosome = proofreading
Polypeptide elongation
Step 2: Peptide bond
Step 3a: Large
subunit shifts
relative to small
subunit and mRNA…
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 + Pi
Polypeptide elongation
07.6-translation_II.mov
Prokaryotes: ~20 aa/sec…
Eukaryotes: ~ 2 aa/sec…
Polyribosomes
Multiple ribosomes
translating one mRNA
5’ to 3’
ECB 7-35
Antibiotics that block prokaryotic protein synthesis