BIOCHEMISTRY scanning mechanism driven by

ATP hydrolysis
EUKARYOTIC TRANSLATION,  Correct AUG is surrounded by
MUTATION AND REPAIR Kozac sequence
 Mrna binds with initiating Met-
trna guided by 5’ cap of mrna
After transcription, mRNA of eukaryotes  Ribosome would find the correct
becomes modified with the addition of the 5’ AUG through the guidance of 5’
cap (7-methylguanosinetriphosphate) and cap and Kozac sequence
poly adenyl tail (consensus sequence)
*AUG: where translation starts
 Forms 48S initiation complex
EUKARYOTIC TRANSLATION - Step 3
 Recruitment of 60S ribosome
- The main features are the same in forming the 80S initiation
prokaryotes and eukaryotes but the complex
details differ  GTP is hydrolyzed
- mRNA of eukaryotes is  Initiation factors are released
characterized by 2 major /dissociated
posttranscriptional modifications  40S +60S =80S
1. 5’ cap  Complex formed is involved in
2. 3’ poly A tail chain elongation

Chain Initiation Chain Elongation

- This is the part of the eukaryotic
translation that is most different from
that of prokaryotes
- Eukaryotes have more initiation
factors involved (EUKARYOTIC
INITIATION FACTORS)
- Characterized by the presence of 13
more initiation factors (eIFs)
- Step 1
 Assembly of 43S preinitiation
complex
 Met-tRNA binds to
ribosomal subunit
 Binding of elongation factor to
such complex
 Different from prokaryotes
because Met-tRNA can already
bind with the ribosome even if
mRNA is not present
 Forms 43S initiation complex
- Step 2
 Recruitment of mRNA
 5’ cap orients the ribosome to
the correct codon AUG via a
- Uses the same mechanism of
peptide formation catalyzed by
peptidyl transferase and ribosome
translocation from different sites as
prokaryotes
- Ribosomes does not contain an E
site
 Main difference vs prokaryotes
 Contains only A and P sites
- Involves two elongation factors
 Addition of amino acid is done
40S with the guidance of the codons
of mRNA
 eEF1: contains 2 subunits
1. eEF1A (counterpart of EF-
Tu)
2. eEF1B (counterpart of EF-
Ts)
 eEF2: counterpart of EF-G,
which causes translocation
Chain Termination
 - ribosomes encounter stop codons
(UAG, UAA, UGA) that are not
recognized by a tRNA molecule
 when stop codons are reached,
release factor would bind such
stop codons
- only one release factor is present in
eukaryotic cells
 catalyzes the hydrolysis of the
bond between the C-terminal
amino acid and the tRNA
 components of protein synthesis
are dissociated
o big and small ribosomal
subunits
o polypeptides
o release factors
= undergo post-translational
processes /modification
- Suppressor tRNA
 A special tRNA that permits
translation to continue through a
stop codon
 Found in cells in which a
mutation has introduced a stop
codon
 Can avoid premature termination
of protein synthesis
 Doesn’t allow continuation of
translation in normal mRNA
New Evidence
- It was believed that translation to
have been physically separated from
transcription, which occurred in the
nucleus
 Research found that the nucleus
has all the components
necessary for translation
(mRNA, ribosomes, protein
factors)
 In isolated test systems, proteins
are translated in the nucleus
 Researchers suggested that
10%-15% of the cell’s protein
synthesis occurs in nucleus
- AUG is not always the start codon
 Peptides that are synthesized to
be presented on the surface of
major histocompatibility
complexes (MHC) use CUG (leu)
as the start codon [Science
Journal, June 2012]
o MHC: surface structure that
carry peptides of viruses
/foreign organisms
o Not clear if such codon
(CUG) may occur in other
proteins other than those
present at the surface of
major histocompatibility
complexes
o Research ongoing
Post-translational Mutation
- what happens to protein when
released from the ribosome
- newly synthesized polypeptides are
frequently modified before they
reach their final form where they
exhibit biological activity
 N-formylmethionine in
prokaryotes is cleaved /removes
o All proteins in prokaryotes
have this bit after protein
production, the polypeptide
will lose its N-fmet
 Specific bonds in precursors are
cleaved. For example:
preproinsulin to proinsulin to
insulin
 Disulfide bonds may be formed
o Also happen between
sulfhydryl groups
o Why end of the preproinsulin
is able to covalently bond
with particular portions of
preproinsulin =functional
insulin has functional
structure with disulfide bonds
- Leader sequence bring to the
particular part of cell to be removed;
not part of a functional protein
- Leader sequences are removed by
specific proteases of the
endoplasmic reticulum; the Golgi
 apparatus then directs the finished
protein to its final destination
 Proteins exported to the other
parts of the cell have leader
sequences at their N-terminal
 Leader sequences direct
proteins to their proper
destination
- Factors such as heme groups may
be attached
 Like in hemoglobin
 Iron (ferric ion) is added to form
the heme group that would
attach to the hemoglobin
 Heme is added to hemoglobin so
that oxygen binds to it
= transported to different parts of
the body
- Amino acids may be modified. For
example, conversions of proline to
hydroxyproline
- Other covalent modifications:
addition of carbohydrates
 Particularly happens in the
formation of antibodies or
immunoglobulins
Proofreading and Repair
- During DNA replication, errors may
be made by RNA polymerase during
the addition of nucleotides
- DNA replication takes place only
once each generation in each cell
- Occur only when cell is going to
divide
 DNA should be replicated with
accuracy to avoid mutations
- DNA polymerase fidelity
 Ability of the polymerase to avoid
or to correct errors in the newly
synthesized DNA strand
 After such proofreading done by
DNA polymerase 1: removes
incorrect nucleotides
immediately after its added to
the growing DNA during
replication
- Proofreading
 Removal of incorrect nucleotides
immediately after they are added
to the growing DNA during
replication
- Errors in hydrogen bonding lead to
errors (mispairing bases) in a
growing DNA chain once in every
104 to 105 base pairs
- Due to proofreading, errors
(spontaneous mutation) only occur
in every 109 to 1010 base pairs
 Proofreading decreases
frequency of errors of DNA
polymerase
MUTATION
- Change in the base sequence of
DNA
- Origins
1. SPONTANEOUS MUTATION
 Occurs during (DNA replication)
normal genetic and metabolic
functions in the cell
o Occurs normally during the
activity of DNA polymerase
when it synthesizes the DNA
 Replication errors: base
mispairing /misreading
 Base modifications caused by
spontaneous hydrolytic reactions
o Ex. base with amino group; a
hydrolytic reaction may
remove the amino group of
that base =erroneously pairs
with a different partner
 Low frequency: 10-9 to 10-10 per
generation
2. INDUCED MUTATION
 Mutagens increase mutation
frequency
 10-3 to 10-4 per generation
 More frequent than spontaneous
Mutagens
 Physical agents
- Ultraviolet light
  Usually affect the part of DNA - Base analogues
with 2 adjacent pyrimidines
(thymine and thymine)
o With no covalent bond
o Covalent bond is formed
between sugar and base
 We are constantly exposed to
UV
 Because of melanin, we are
protected from effects of UC
 What happens when UV hits our
DNA
o Causes covalent bond
between the bases
o Cyclobutene ring is formed
= distorts DNA conformation
= causes error in replication

- Ionizing radiation
 Radicals can be formed by
chemicals (antioxidants)
 X-rays, gamma rays, radiation
 Interacts with H2O to form OH
radical
 OH radical reacts with H in DNA
 DNA radical is formed
 Chain reactions
 DNA breaks
 When x-ray hits our cells, it
strikes water
o Water is ionized into h and
OH radicals
 Radicals are reactive
o May easily react to other
molecules
o Ex. OH radical easily reacts
with H which is abundant in
DNA; easily forming water
and the radical transfers to
DNA; now the DNA is
radical /reactive until it
breaks and causes error in
replication
= single strand/ mutation

 Chemical Agents
- Nitrous acid
- Intercalating agents
- 5-bromouracil