Basic Principles of Medicine 1

Module: Foundations to Medicine

Lecture No: 14

Lecture Title:
Translation and Post-translational
Modification

Name of Lecturer: Dr Felicia Ikolo

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Assigned Reading

• Lippincott’s Biochemistry: Chapter 32

• Concept map: page 462

• Questions: 32.1-32.3, 32.5-32.9

 Additional Resources

YouTube videos:

• The big picture of translation:

• http://www.youtube.com/watch?v=5bLEDd-PSTQ
• 3 minutes 32 seconds.

• Interpreting the genetic code:

• https://www.youtube.com/watch?v=-Ht81lHiJac
• 4 minutes 16 seconds.
 Objectives (2 lectures)
SOM.1a.BPM1.1.FTM.3.BCHM.MB.1101 Explain why the genetic code is collinear with DNA, triplet in nature, redundant
(degenerate), and non-overlapping.

Video 1
SOM.1a.BPM1.1.FTM.3.BCHM.MB.1102 Define the terms: reading frame and frame-shift mutation.

Video 2
SOM.1a.BPM1.1.FTM.3.BCHM.MB.1103 Translate and interpret the standard genetic code table and be able to identify the

Videos 1 & 2
initiation codon as well as the 3 terminator codons.

SOM.1a.BPM1.1.FTM.3.BCHM.MB.1104 Explain the universal nature of the genetic code (with minor differences in

Viseos 1 & 2
mitochondria) and predict the sequence of a peptide if the coding strand of the DNA is
provided.

SOM.1a.BPM1.1.FTM.3.BCHM.MB.1105 Discuss how protein synthesis proceeds from the N-terminal to their C-terminal ends
and explain how this creates a naming convention for peptides.

Video 1
SOM.1a.BPM1.1.FTM.3.BCHM.MB.1106 List and describe the major function of the major types of RNA in cells, including

Video 2
mRNA, tRNA, rRNA, snRNA, snoRNA, miRNA.

SOM.1a.BPM1.1.FTM.3.BCHM.MB.1107 Describe the subunit composition and the basic structure of prokaryotic 70S and

Video 3
eukaryotic 80S ribosomes, including the basis for their names and be able to identify
5
the A site, the P site, and the E site on the ribosome.
 Objectives: continued
SOM.1a.BPM1.1.FTM.3.BCHM.MB.1108 Describe the structure, function, and charging of tRNAs.

Video 3
SOM.1a.BPM1.1.FTM.3.BCHM.MB.1109 Describe the codon/anticodon interaction and discuss the wobble hypothesis.

Video 3
SOM.1a.BPM1.1.FTM.3.BCHM.MB.1201 Describe the sequence of events that occurs during translation in prokaryotes
(initiation, elongation and termination) and list the major differences between
prokaryotic and eukaryotic translation.

Lecture
SOM.1a.BPM1.1.FTM.3.BCHM.MB.1202 Explain how diphtheria toxin interferes with eukaryotic translation and explain the
mode of action of common antibiotics that interfere with translation: Initiation
inhibitors (streptomycin), Elongation inhibitors (Tetracycline, chloramphenicol,

Lecture
erythromycin, puromycin, cycloheximide).

SOM.1a.BPM1.1.FTM.3.BCHM.MB.1203 List and review the major types of post-translational modifications: Zymogen
activation (trypsinogen → trypsin as example), Serine/threonine phosphorylation
(regulation of enzymes in metabolism), Tyrosine phosphorylation (Insulin receptor as

Lecture
an example), O-linked glycosylation, N-linked glycosylation and Lipid anchoring (e.g.
farnesyl groups to RAS).
SOM.1a.BPM1.1.FTM.3.BCHM.MB.1204 Describe proteolytic processing using insulin as an example.

Lecture
6
Translation: Formation of a polypeptide
polymer from an RNA template

1. Activation of the monomer

2. Initiation
3. Elongation
4. Termination
5. Processing the polymer

We will describe translation in prokaryotes, and provide notes

regarding the eukaryotic system when appropriate
7
 Amino acids are activated
by attachment to tRNA

• Charging the tRNA is a two step

process:
1. Enzyme bound amino-acid-adenylate
2. Formation of the aminoacyl-tRNA

• Reaction is driven by hydrolysis of

pyrophosphate

• tRNA attached to amino acid is

called the “charged tRNA”
8
(Charged tRNA)
 fMet-tRNA
• N-formylmethionine as the first
amino acid – in prokaryotes &
in mitochondria.

• This special tRNA (fMet-tRNA) is

recognized differently by the
ribosome – allows initiation

• Prokaryotes have 2 tRNAs for

methionine, one allows
formation of fMet, the other
recognizes internal AUG codons
Use of Met-tRNA-MET as 1st tRNA

Prokaryotes have 2 tRNA that recognize AUG

Prokaryotes
& mitochondria
of eukaryotes Formylated MET for Normal Met-tRNA for internal codons
first codon

Eukaryotes also have 2 tRNA that recognize AUG

Eukaryotes The first codon also uses Normal Met-tRNA for internal codons
MET, and it has a special
tRNA for this first codon
(but the MET amino acid is
not formylated)

10
The idea is to get the first MET containing tRNA into the P-site to allow initiation
Correct alignment of the AUG codon with
respect to the ribosome

• Prokaryotes: Shine Dalgarno sequence is purine rich and resides

a few (5-10) bases 5’ to the start codon
• Eukaryotes: lack Shine Dalgarno sequence, therefore eukaryotic
small ribosome binds close to the cap at the 5’ end, scans until it
encounters the AUG start codon.
Protein
Synthesis
Catalyzed by an enzymatic activity of the RNA
portion of the 50S ribosome – a Ribozyme
 Protein
Synthesis
-continued
• EF-G is the
prokaryotic
protein

• In
eukaryotes,
EF-G is
called EF-2
Termination codons are UAA, UAG, & UGA
Termination is more easily understood in Eukaryotes

• Stop codon has no tRNA

• Stop codon causes elongation to stall

• Peptide is held in the P-site attached to the tRNA

• Release Factor (RF) diffuses in to the A-site

• RF allows peptidyl transferase to cleave ester bond between

tRNA and the peptide
• GTP dependent activity

• After release of the peptide, the ribosome dissociates into its

subunits which may then begin a new round of translation

15
 Electron microscopy showing coupled
transcription and translation in prokaryotes

DNA

Short transcripts as Polyribosome formation longer transcripts as

RNA polymerase on the RNA transcript RNA polymerase nears
begins transcription the end of the gene
20
Proteins are not labeled, so we don’t see them
A polyribosome: ribosomes
simultaneously translating one mRNA
Protein folding occurs during translation:
spontaneously or facilitated by chaperones

Chaperones ensure that only a limited number of

folds are available to a newly synthesized protein. 22
Some major differences between Prokaryotic
& Eukaryotic Translation

In Eukaryotes, the first MET is not formylated

Prokaryotes – polycistronic Typically, methionine is NOT found as

Eukaryotes – monocistronic the first amino acid on a mature protein
– post translationally modified

Prokaryotes – may select an internal AUG site to start translation

Eukaryotes – typically start translation at first AUG site

Prokaryotes – translation and transcription are coupled

Eukaryotes – may not couple because of nuclear membrane
23
➢After the nucleic acid discussions, you should be able to
identify the 5` and 3` ends, as well as determine the
complementary sequence of a nucleic acid:

GGACGTATCCTGGAC
GGACGUAGUAACUGA

➢Which of the above strands is DNA? Which is RNA?

➢Write the complementary sequence of the DNA

➢Using the mRNA strand, predict the amino acid

sequence after translation
 Compounds affecting protein synthesis
• Diphtheria toxin inactivation of EF-2 by ADP-ribosylation

• Antibiotic Inhibition of initiation

• Streptomycin (aminoglycoside)
• Prevents assembly of ribosome (binds to 30s subunit)

• Antibiotic Inhibition of elongation

• Tetracycline - four (tetra) ring (cyclic) structure
• Block elongation by preventing aminoacyl-tRNA access to the A-site
• Erythromycin (macrolide)
• Binds to the 50S subunit of the complete (70S) ribosome
• Blocks ribosome translocation
• Chloramphenicol
• Inhibits peptidyl transferase activity in prokaryotes
• At high levels, may inhibit mitochondrial translation
• Cycloheximide
• Inhibits eukaryotic peptidyl transferase activity
• Puromycin
• Causes premature termination of translation in both prokaryotes and
25
eukaryotes There are many more
 nhibitors of
Protein
Synthesis
Inhibitors of
Protein
Synthesis
-continued

• EF-G is the
prokaryotic
protein

• In eukaryotes,
EF-G is called
EF-2
 Post-translational Modifications
• May occur as the polypeptide is being translated
• May occur after translation is completed
• Many types – we will discuss a few:
• Zymogen activation
• Ser/Thr phosphorylation
• Tyr phosphorylation
• O - linked glycosylation
• N - linked glycosylation
• Lipid anchoring

• There are many more!

28
Zymogen Activation
• Zymogen (or proenzyme): an inactive enzyme precursor
• Activated within an organism into active enzymes
• Activation by enzymatic cleavage of peptide bonds of the
zymogen molecule
• Cascade of zymogen activation:
• Caspases to activate apoptosis
• blood coagulation
• Digestion of proteins
Protein phosphorylation by a kinase
• Serine/Threonine
• Phosphate is transferred from ATP to a protein on a Ser /
Thr residue of a protein
• More common than tyr phosphorylation

Involved in peptide bond

in the protein

30
Protein phosphorylation by a kinase
• Tyrosine
• Phosphate is transferred from ATP to a protein on
a Tyr residue of a protein
• The Insulin Receptor (IR) is a tyrosine kinase

31
Glycosylation: may occur as either one type of
linkage or with both on the same protein
• O – linked
• Glycosylation on the OH group
of Ser/Thr
• Often found as extracellular
proteins or as membrane
bound proteins
• Glycan groups always face
extracellular side

• N – Linked
• Glycosylation on the Asn
residue (not Gln)
• Either of two types (high
33
mannose, or complex)
 Lipid Anchoring

• Farnesyl is a 15
carbon
isoprenoid group
which may be
attached to
cysteine.
 Extracellular
The cell targets Ras to the
plasma membrane by a lipid
anchor mechanism

Lipid anchoring: Acts to

anchor Ras to inner
leaflet of plasma
membrane

S
Ras
Protein

Cytosol
35
 Proteolytic processing of insulin

•Insulin and its C-peptide are packaged together into secretory

vesicles to be made ready for secretion
•C-peptide is essential for proper insulin folding
•C-peptide is a good indicator of insulin production and secretion
because its (C-peptide) half-life in the plasma is longer than that36of
insulin.
Proteins destined for secretion are translated into the rER
Plasma membrane

Signal
sequence

3. Translation of the polypeptide is directed 4. The signal sequence is cleaved in the

into the lumen of the rER and forms lumen of the rER – forms “proinsulin”
“preproinsulin” 37
 Maturation of insulin occurs in the golgi

Insulin is secreted by the β-cell in response to high blood glucose38

 Insulin is derived from a single polypeptide

•A chain = 21 amino acids, B chain = 30 amino acids

•Insulin and the C-peptide are released into the blood
•Insulin binds to Insulin Receptor (tyrosine kinase) to signal that blood
glucose is high
•There might be (?) a cellular role for the C-peptide
•Measurement of the C-peptide levels has a clinical value (discussed
after the midterm) 39