1. Protein Synthesis Kim Foreman, PhD kforema@luc.edu
Cancer Center, Room 235
August 10, 2018

2. Decoding mRNA ?
mRNA
proteins

3. Translation A A G U C A G U C Messenger RNA mRNA Codon Translation
Polypeptide
(amino acid
sequence)
Protein
Lysine
Serine
Valine

4. The Genetic Code

5. The Genetic Code
The genetic code is universal All known organisms use the same genetic code
The genetic code is degenerate Multiple codons encode the same amino acid e.g. GGU GGC, GGA, and GGG all encode for glycine Degeneracy is mostly at the third base of the codon (Wobble base)
Some codons have additional functions
AUG encodes methionine Methionine can be used within a protein sequence and is generally the first amino acid at the beginning of the polypeptide chain UAA UAG and UGA do not encode for an amino acid These are stop codons that terminate protein synthesis

6. An mRNA consisting of contiguous triplet codons can be read in three different reading frames.

7. Steps of Translation Charging of the tRNA Initiation Elongation
Termination

8. Transfer RNA

9. Wobble permits some tRNAs to recognize more than one codon

10. Reactions catalyzed by aminoacyl-tRNA synthetases
High energy
ester linkage
Eukaryotes: different synthetase of each amino acid
Prokaryotes: one synthetase couples more than one amino acid

11. Reactions catalyzed by aminoacyl-tRNA synthetases

12. Interaction of a tRNA with its aminoacyl-tRNA synthetase
Amino acid
accepting arm
ATP
tRNA
Aminoacyl-
tRNA synthetase
Anticodon
region

13. Aminoacyl-tRNA synthetase must recognize the correct tRNA
The anticodon is directly recognized by
the synthetase through three adjacent
binding pockets that are complementary
to the shape and charge of the nucleotide
in the anticodon.
For amino acids with multiple
unrelated anticodons, segments in the
acceptor stem and elsewhere
are critical.
acceptor stem
for the amino acid
Site(s) in a tRNA used by one or more
aminoacyl-tRNA synthetases to recognize
their cognate tRNA.
anticodon

14. Aminoacyl tRNA synthetase must select the correct amino acid
correct amino acid has the highest affinity for the synthesis site
proof-reading prevents the addition of the wrong amino acid.
accuracy of tRNA charging is 1 mistake in 40,000 couplings

15. Translation initiation in prokaryotes
Prokaryotic mRNA
Initiation occurs at AUG codons with properly spaced Shine-Dalgarno sequence
5’ AUG SD AUG AUG SD AUG ’
initiation codon with
Shine-Dalgarno site
internal Met codon
does not have
Shine-Dalgarno site
initiation codon with
Shine-Dalgarno site
Shine-Dalgarno (SD) site consists of 3-9 contiguous bases in the mRNA that base
pair with the 3’ end of 16S rRNA and is located ~5 bases upstream of the initiator codon

16. GAUUCCUAGGAGGUUUGACCUAUG CGA GCU
The Shine-Dalgarno sequence functions as a binding site for the bacterial 16S ribosomal subunit
5’ ’
AGCACGAGGGGAAAUCUGAUGGAACGCUAC E. coli trpA
UUUGGAUGGAGUGAAACGAUGGCGAUUGCA E. coli araB
GUAAACCAGGUAACAACCAUGCGAGUGUUG E. coli thrA
CAAUUCAGGGUGGUGAAUGUGAAACCAGUA E. coli lacl
AUGUACUAAGGAGGUUGUAUGGAACAACGC phage cro
Pairs with Pairs with
16S rRNA initiator tRNA
GAUUCCUAGGAGGUUUGACCUAUG CGA GCU 3’ 5’
UCCUCC
mRNA U A C G
16S rRNA
Shine-Dalgarno
sequence

17. synthesis in prokaryotes
Initiation of protein
synthesis in prokaryotes
The 16S rRNA subunit base pairs with the Shine-Dalgarno
sequence immediately upstream from the initiating AUG codon. This positions the initiating codon in the ribosome.
Because the ribosome assembles on the start codon, bacterial mRNA can be polycistronic.
Translation initiation factors (IF1, IF2, IF3) orchestrate this
interaction and assembly of the large ribosomal
subunit to form the functional ribosome.
Small ribosomal subunit provides the framework to
accurately match the codon/anticodon while the large
ribosomal subunit catalyzes the peptide bond formation
In prokaryotes, translation begins with a special
tRNA that carries a modified methionine-
formylmethionine.

18. synthesis in eukaryotes
Initiation of protein
synthesis in eukaryotes
Initiation tRNA (with bound eIF-2-GTP) is loaded into
the small ribosomal subunit.
eIF-4E and eIF-4G bind to both the 5’ cap and the
poly A tail of the mRNA to ensure it is intact.
The small ribosomal subunit binds to the mRNA by
recognizing the cap and its associated eIFs.
The small ribosomal subunit scans for the first AUG.
eIF-2-GTP is hydrolyzed, eIFs dissociate and the large
ribosomal subunit binds to the small subunit.
The initiation tRNA is in the P-site of the ribosome.
Protein synthesis is ready to begin.

19. Reactivation of eIF2-GDP
eIF2+GTP eIF2-GDP during met-tRNA transfer
eIF2-GDP must be recycled to eIF2-GTP .
Exchange is catalyzed by eIF2B (guanine nucleotide
exchange factor)
Reactivation of eIF2 is catalyzed by eIF2B
eIF2-GDP + GTP  eIF2-GTP + GDP

20. synthesis in eukaryotes
Elongation of protein
synthesis in eukaryotes
Ribosome contains four binding sites
mRNA site, A-site, P-site, E-site
At the end of initiation, the initiation tRNA is in the P-site
of the ribosome.
A tRNA enters the A-site and base pairs with the codon
of the mRNA. A peptide bond is formed between the
amino acids in the A and the P-site.
The peptidyl transferase catalytic activity of the large
ribosomal subunit is accompanied by conformational
changes that shift the two tRNAs into the E- and P-
sites. Additional conformational changes move the mRNA
3 nucleotides so the ribosome is reset and ready
for the next tRNA.

22. Elongation requires GTP hydrolysis
Two elongation factors (EF-Tu and EF-G in bacteria;
EF-1, EF-2 in eukaryotic cells).
EF-Tu+GTP binds to a charged tRNA as it enters the
ribosome. GTP hydrolysis and the exit of the EF-Tu from the ribosome cause two short lags that provide an opportunity for an incorrectly bound tRNA to exit the ribosome prior to incorporation into the polypeptide chain (responsible for 99.9% accuracy of the ribosome
in translation)
EF-G+GTP binds near the A-site and accelerates the
movement of tRNAs to the P- and E-sites.
The GTP hydrolysis catalyzes the conformational
change that is needed to move the tRNA and advance
the mRNA forward by one codon.

23. Termination of translation
When a stop codon is reached, release factors bind
to the ribosome and forces the peptidyl transferase
of the ribosome to catalyze the addition of a water
molecule instead of an amino acid. This frees the
carboxyl terminus from the tRNA and the
completed protein is released.
Nakamura and ITO 2003, TIBS 28:99

24. Fidelity of protein synthesis
(1) Aminoacyl tRNA synthetase must recognize the correct tRNA
(2) Aminoacyl tRNA synthetase must select the correct amino acid
(3) mRNA must be fully processed (in eukaryotes) prior to translation initiation
(4) The ribosome matches the mRNA codon to the tRNA anticodon.
The correct anticodon forms a stronger interaction with the codon than an
incorrect pairing.
(5) GTP hydrolysis and release of EF-Tu elongation factor provide short
delays allowing the tRNA to be released from the A-site of the ribosome
before an incorrect amino acid is irreversibly added into the peptide chain.

25. Differences in transcription and translation between prokaryotes and eukaryotes

26. Differences in transcription and translation between prokaryotes and eukaryotes

27. Comparison of a prokaryotic and eukaryotic ribosome

28. Puromycin – mimics tRNA
“molecular mimicry”

29. Puromycin – an example of molecular mimicry

30. Creating a functional protein
Folding
Binding required co-factors
Covalent modifications
Assemble with any partner proteins

31. Proteins can begin to fold as they are synthesized

32. Molecular chaperones can assist in protein folding
hsp70
Interact with exposed hydrophobic
patches on proteins immediately
after leaving the ribsome.
ATP hydrolysis causes the hsp70 to
bind tightly to the protein. Repeated
binding and release help protein refold.
hsp60
Forms a barrel shaped structure
to isolate misfolded proteins.
This prevents their aggregation
and provides a favorable
environment for them to
refold.