Molecular biology Dr. Mohammed Albayati 2015
Protein Synthesis and Targeting
Codon–anticodon interaction In the cleft of the ribosome, an antiparallel
formation of three base pairs occurs between the codon on the mRNA and
the anticodon on the tRNA. If the 5’ anticodon base is modified, the tRNA
can usually interact with more than one codon.
Wobble The wobble hypothesis describes the nonstandard base pairs that
can form between modified 5’anticodon bases and 3’codon bases. As long
as the distances between the ribose units were close to normal.
1. No purine–purine or pyrimidine–pyrimidine base pairs are allowed as
the ribose distances would be incorrect.
2. No single tRNA could recognize more than three codons, hence, at
least 32 tRNAs would be needed to decode the 61 codons, excluding
stop codons.
3. tRNAs can recognize either one, two or three codons, depending on
their wobble base (the 5’anticodon base).
a) If it is C it will recognize only the codon ending in G as usual.
b) If it is G, it will recognize the two codons ending in U or C.
c) If U, which is subsequently modified, it will pair with either A or G.
d) The wobble nucleoside is never A, as this is converted to inosine
which then pairs with A, C or U.
Shine–Dalgarno sequence The ribosome binding site is a sequence just
upstream of the initiation codon in prokaryotic mRNA which base-pairs
with a complementary sequence near the 3’end of the 16S rRNA to position
the ribosome for initiation of protein synthesis.
Polysomes
Polyribosomes (polysomes) form on an mRNA when successive ribosomes
attach, begin translating and move along the mRNA. A polysome is a
complex of multiple ribosomes in various stages of translation on one
mRNA molecule.
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Molecular biology Dr. Mohammed Albayati 2015
Initiator RNA A special tRNA (initiator tRNA), recognizing the AUG start
codon, is used to initiate protein synthesis in both prokaryotes and
eukaryotes. In prokaryotes, the initiator tRNA is first charged with
methionine by methionyl-tRNA synthetase. The methionine residue is then
converted to N-formylmethionine. In eukaryotes, the methionine on the
initiator tRNA is not modified. This Initiator RNA differs from the tRNA
that inserts internal Met residues.
MECHANISM OF PROTEIN SYNTHESIS
There are three stages of protein synthesis:
 initiation – the assembly of a ribosome on an mRNA;
 elongation – repeated cycles of amino acid delivery, peptide bond
formation and movement along the mRNA (translocation);
 termination – the release of the polypeptide chain.





Schematic representation of eukaryotic ribosome and the sites involved in
protein synthesis. P (peptidyl) site initially binds initiator tRNA, later binds
growing peptide chain. A (aminoacyl) site binds incoming tRNA molecule
with activated amino acid (ie, bound amino acid with high-energy bond). E
site receives uncharged tRNA once amino acid has been added to
polypeptide.
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Molecular biology Dr. Mohammed Albayati 2015
 initiation In prokaryotes, initiation requires the large and small
ribosome subunits, the mRNA, the initiator tRNA, three initiation
factors (IFs) and GTP.
IF1 and IF3 bind to the 30S subunit and prevent the large subunit
binding. IF2 + GTP can then bind and will help the initiator tRNA to
bind later. This small subunit complex can now attach to an mRNA via
its ribosome-binding site. The initiator tRNA can then base-pair with
the AUG initiation codon which releases IF3, thus creating the 30S
initiation complex. The large subunit then binds, displacing IF1 and
IF2 + GDP, giving the 70S initiation complex which is the fully
assembled ribosome at the correct position on the mRNA.
 Elongation involves the three factors (EFs), EF-Tu, EF-Ts and EF-G,
GTP, charged tRNAs and the 70S initiation complex (or its
equivalent).
o It takes place in three steps.
o A charged tRNA is delivered as a complex with EF-Tu and GTP. The
GTP is hydrolyzed and EF-Tu_GDP is released
o Peptidyl transferase makes a peptide bond by joining the two
adjacent amino acids without the input of more energy.
o Translocase (EF-G), with energy from GTP, moves the ribosome one
codon along the mRNA, ejecting the uncharged tRNA and
transferring the growing peptide chain to the P-site.
 Termination Release factors (RF1 or RF2) recognize the stop codons
and, helped by RF3, make peptidyl transferase join the polypeptide
chain to a water molecule, thus releasing it. Ribosome release factor
helps to dissociate the ribosome subunits from the mRNA.
 In eukaryotes a single release factor, eRF, recognizes all three stop
codons and requires GTP for activity.
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Molecular biology Dr. Mohammed Albayati 2015
Many antibiotics inhibit the protein synthesis at some specific
steps:
Streptomycin: It is a highly basic trisaccharide.
It interferes with the binding of f-met t-RNA to ribosomes and
thereby inhibits the initiation process.
It also leads to misreading of m-RNA.
Puromycin: This inhibits protein synthesis by releasing nascent
polypeptide chains before their synthesis is complete. It binds to the
A site on ribosome and inhibits the entry of aminoacyl-t RNA. It acts
both in bacterial and mammalian cells.
Tetracycline: It binds to the 30S subunit and inhibits binding of
aminoacyl t-RNA, thus inhibits the initiation process.
Chloramphenicol: It inhibits the peptidyl transferase activity of 50S
subunit. Thus it inhibits the process of elongation.
Cycloheximide: This inhibits peptidyl transferase activity of 60S
ribosomal subunit in eukaryotes. It also inhibits elongation.
Erythromycin: It binds to the 50S subunit and inhibits translocation.
Diphtheria toxin: Corynebacterium diphtheriae produces a lethal
protein toxin. It binds with EF-2 in eukaryotes and blocks its
capacity to carry out translocation.
TRANSLATIONAL
CONTROL
AND
POST-
TRANSLATIONAL EVENTS
In prokaryotes, the level of translation of different cistrons can be affected
by:
(i) the binding of short antisense molecules, (ii) the relative stability to
nucleases of parts of the polycistronic mRNA, and (iii) the binding of
proteins that prevent ribosome access.
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Molecular biology Dr. Mohammed Albayati 2015
In eukaryotes, protein binding can also mask the mRNA and prevent
translation, and repeats of the sequence 5’-AUUUA-3’ can make the mRNA
unstable and less frequently translated.
A specific and interesting example is how body regulates the amount of
iron in cells, iron being essential for the activity of some proteins, but
harmful in excess. Iron is transported into cells by the transferrin receptor
protein and is stored within cells bound to the storage protein ferritin.
The mRNA for each of these proteins contains noncoding sequences that
can form stem-loop structures, called the iron response element (IRE)
to which an iron sensing protein (ISP) can bind. However, the position of
the IRE and the action of the bound ISP is very different. In the transferrin
receptor mRNA, the IRE is in the 3’ noncoding region and when the ISP
binds, which it does when iron is low, it stabilizes the mRNA and allows
more translation. But when iron levels are high the ISP dissociates from the
IRE and unmasks destabilizing sequences that are then attacked by
nucleases, thus reducing translation due to mRNA degradation.
When iron levels are high, not only is the transferrin mRNA
destroyed, but the translation of ferritin storage protein mRNA is increased.
This occurs because at low iron levels the IRE, which is located in the 5’
noncoding region, binds ISP which in turn reduces the ribosome's ability to
translate the ferritin mRNA. When iron levels rise, the ISP again dissociates
from the IRE, but in this case causes an increase in translation of ferritin
because the ribosome's progress is not repressed. This translational control
system rapidly and responsively regulates intracellular iron levels. There are
other examples of translational control that work via protein binding in the
vicinity of destabilizing sequences and inhibiting them.
Other examples of translational control include micro RNAs in eukaryotes
that bind to mRNAs to which they are complementary, and either cause
degradation or translational repression of the mRNA
Polyproteins A single translation product that is cleaved to generate two or
more separate proteins is called a polyprotein. Many viruses produce
polyproteins.
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Molecular biology Dr. Mohammed Albayati 2015
Protein targeting
Certain short peptide sequences in proteins determine the cellular location
of the protein, such as nucleus or mitochondrion. The signal sequence of
secreted proteins causes the translating ribosome to bind factors that make
the ribosome dock with a membrane and transfer the protein through the
membrane as it is synthesized. Usually the signal sequence is then cleaved
off by signal peptidase.
Protein modification the most common alterations to nascent polypeptides
are those of cleavage and chemical modification.
Cleavage examples are
a) Signal sequences are also usually cleaved off secreted proteins
b) Release mature fragments from polyproteins eg. Ubiquitin is made
as a polyprotein containing multiple copies linked end-to-end, and
this must be cleaved to generate the individual ubiquitin molecules,
c) Remove internal peptides as in the case of insulin
d) Trimming both N- and C-termini.
There are many chemical modifications that can take place on all but six of
the amino acid side chains, ( Ala, Gly, Ile, Leu, Met and Val. )The
modifications include acetylation, hydroxylation, phosphorylation,
methylation, glycosylation and even the addition of nucleotides.
Hydroxylation of Pro is common in collagen, and some of the histone
proteins are often acetylated. Often phosphorylation controls the activity
of the protein.
Protein degradation
Different proteins have very different half-lives. Regulatory proteins tend to
turn over rapidly and cells must be able to dispose of faulty and damaged
proteins, modified or inherently unstable proteins, these are marked for
degradation by having multiple molecules of ubiquitin covalently attached.
The ubiquitinylated protein is then degraded by a 26S protease complex.
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Molecular biology Dr. Mohammed Albayati 2015
The majority of the degraded proteins are reduced to amino acids that can
be used to make new proteins, but random peptide fragments of nine
amino acids in length are attached to peptide receptors (called major
histocompatibility complex class I molecules) and displayed on the surface
of the cell. Cells display around 10 000 of these peptide fragments on their
surface and this gives them a unique identity since different individuals
display a different set of these peptide fragments on the surface of their
cells. These differences explain, not only why transplanted organs are often
rejected, but also why cells infected by viruses (which will display foreign
peptide fragments on their surfaces) will be destroyed by the immune
system.
In eukaryotes, it has been discovered that the N-terminal residue
plays a critical role in inherent stability.
 8 N-terminal aa correlate with stability:
Ala Cys Gly Met Pro Ser Thr Val
 8 N-terminal aa correlate with short t1/2:
Arg His Ile Leu Lys Phe Trp Tyr
 4 N-terminal aa destabilizing following chemical modification:
Asn Asp Gln Glu
Methods of Regulation of Gene Expression in Eukaryotes
1. RNA processing:
2. Gene amplification: the expression of a gene is increased several-fold.
This is commonly observed during the developmental stages of eukaryotic
organisms.
In pateints receiving Methotrexate therapy malignant cells can
develop drug resistance by increasing the number of genes for the
enzyme dihydrofolate reductase.
3. Gene rearrangement eg. synthesis of light chains of immunoglobulins
(Igs).
4. Gene regulation by histones and nonhistone proteins:
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Molecular biology Dr. Mohammed Albayati 2015
post-translational modifications of the different histones. Such
modified histones can regulate gene expression.
5. Class switching: In this process, one gene is switched off and a closely
related gene takes up the function.
Examples: Class switching is best illustrated by Hb
Zeta-eta → α γ → then α β and α δ
6. Binding of regulatory proteins to DNA:
• Helix-turn helix
• Zinc finger motif, and
• Leucine zipper motif.
7. Role of enhancers and silencers
Figures and diagrams are present in the ppt of these lectures
This will conclude our lectures for this year
You are expected to read more from your text book
And your lecture hand notes to pass the final exam.
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