this is now the last part of our

lectures in nucleic acids

we will discuss here eukaryotic
translation
mutation and repair
so you recall that in
the transcription after the
transcription of the eukaryotic
gene the mrna
becomes modified with the addition of a
five prime cup
and which is made up of the uh
seven methyl guanosine triphosphate
cup at the five prime end and the
addition of a poly tail
only a tail or polyadeny adenyl
tail
the main features of translation are the
same
in prokaryotes and eukaryotes
but the details differ
in chain initiation
most of the differences are seen
between eukaryotes and prokaryotes
in eukaryotic translation there are more
initiation factors that are involved
which also also
referred to as eukaryotic initiation
factors
or e i f so differentiate this from
prokaryotic initiation factors in that
you have a
small letter e here to denote eukaryotic
this initiation occurs in three steps
first is the assembly
of a pre 43s pre-initiation complex
this involves the binding of
methionine prna
to the small subunit of the ribosome
which is 40s
40s ribosomal subunit
there is also the binding of elongation
factors
to this complex you will note that this
is different from prokaryote in that
m method trna may already bind with the
ribosome even if the
messenger rna is not yet
present
the second step would be the recruitment
of the messenger rna
the messenger rna now binds with the
initiating meth trna
this is guided by the
pipe prime cup of the mrna
the ribosome would find the correct aug
through the guidance of the five prime
cup
and uh this is also guided by the
presence of
the consensus sequence which is called
the cossack sequence
if there is an aug before this
but is not surrounded by a cossack
sequence
that aug may be skipped
by the ribosome and would start
translation
at the correct aug
so here the the complex form
is now called the 48 s initiation
complex this is followed by
the recruitment of the 60s
ribosomal subunit so that's the big
ribosomal subunits of eukaryote the 60s
to form an ats initiation
complex so when 40s and 60s ribosomal
subunits are associated together
they become ats ribosomes
at this point gtp is hydrolyzed
and the initiation factors are
released or dissociated from the complex
this complex now would be involved in
the next
stage which is the elongation of the
polypeptide the same mechanisms
in of peptide bond formation
catalyzed by the peptidyl transferase
and ribosome translocation
from the different sites as in
prokaryotes the difference is that
the ribosome does not have an e site
the ribosome only contains the a
and the p side so that is true for the
eukaryote ribosomes
and and other factors protein factors
are involved here this time they are the
elongation
factors so the ef ef1
and ef2 and
once again there's a left small letter e
before ef to denote that these are
eukaryotic
elongation factors so a series
of addition of amino acids would be
made but as guided by the
codons of the mrna
finally when the stop codons are reached
the a release factor would bind
these stop codons note that
only one release factor is present
in eukaryotic cells and it is able to
bind
to all of the three stop codons this
release factor
catalyzes the hydrolysis of the bond
between the c
terminal amino acid and the t
rna thus the components
of the
protein synthesis are dissociated the
big and the small
ribosomal subunits are dissociated the
polypeptide is dissociated
along with the release factors
the polypeptide now will
be will undergo some post-translation
modification
um in eukaryotic cells
there is one special type of prna which
is called
the suppressor trna it permits
translation to continue
even if there is a stop codon
this stop codon may have been produced
or
introduced because of a mutation
therefore this can avoid premature
termination of
protein synthesis
but this suppressor trna
would not work or would not allow
continuation of translation
in a normal mrna
there are new evidences that would
change our concept
about transcription and translation
it is believed that translation have
been
physically separated from transcription
because in eukaryotes because
of the presence of the nucleus
transcription occurs in the nucleus
while translation occurs in the
cytoplasm
but new evidence has been found
in some studies
that show that the nucleus has all the
components necessary for transcript
translation there is an n there is
mrna there are ribosomes and protein
factors in the nucleus
and and when they uh
experimented on isolated test systems
they found that proteins are translated
in the nucleus
and this researcher suggested that 10 to
15
of the cells protein synthesis occurs
in the nucleus so the old
concept that translation always occurs
in the cytoplasm is no longer true
in addition it's also found that
aug is not always the start codon
peptides that are synthesized to be
presented
on the surface of major
histocompatibility complexes
use cug rather than aug as the start
codon
ncu g codes for leucine
this was a finding by the researchers in
2012 and was published in science
in the journal science
it is not clear whether such
codon initiating codon also
may occur in other proteins other than
those that would be
present on the surface of major
histocompatibility complex
so research is still
ongoing
so what happens to the protein when
it is released from the ribosomes
the newly synthesized polypeptides are
frequently modified
before they reach the their final form
in which they exhibit biologic activity
so there are changes or modifications
that occur
with the polypeptide one of these
is the removal of meal methionine
in prokaryotes you'll find that
not all proteins of in prokaryotes have
n-4 meal methionine after
protein production the polypeptide
will lose its n-4 meal with bioneed
another is that specific bonds in
precursors
are clean as what happens with
pre-pro insulin that uh transform into
pro-insulin
and then to insulin
there's also disulfide bonds that may be
formed
in these polypeptides so in this example
you have insulin
this is the precursor of insulin
which would undergo cleavage of some of
some segments uh particularly
the end segment of the pre-pro insulin
to form
the pre-pro insulin
the next cleavage occurs here
and here such that this central segment
is cleaved and what would be left would
be
this one and you also note that
disulfide bonds are formed between
sulfhydryl groups that's why
this particular end of the prepro
insulin
is able to covalently bind with
this particular portion of the prepro
insulin so that finally the functional
insulin has this structure with
disulfide bonds
so that's one good example of how
the polypeptide is
modified to become functional
other modifications include the removal
of what are called
leader sequences leader sequences
are usually present on the
n-terminal of proteins
these leader sequences lead
the protein to their proper destination
but these sequences are later removed
by specific proteases that are found in
the endoplasmic reticulum
finally when these are removed
the golgi apparatus directs the finished
protein
to its final destination
a hem group may be attached to the
protein just like
in hemoglobin and iron
ion iron no ferric ion
is added to form the heme group it's
attached to
the hemoglobin
or in some cases the amino acids may be
modified
as in the conversion of proline to
hydroxy
proline
there may be also addition of other
groups such as carbohydrates
this particularly happens
in the formation of antibodies or
immunoglobulins but there are other
proteins that may also have
added carbohydrates these are the
modifications that occur
to the polypeptide after
synthesis
now let's talk about mutation
you may refer to pages 282 to 287 of
campbell biochemistry
with regards to the features of this
mutation
let us recall that during dna
replication
errors may be committed by the dna
polymerase during
addition of nucleotides and also recall
that
dna replication takes place only once
in each generation in its cell
as we we have mentioned before dna
replication will
occur only when the cell is going
to divide if it is not dividing it will
not replicate
its dna
and it is important that the dna is
replicated with
accuracy to avoid mutation
and this accuracy is related to what is
called dna polymerase
fidelity the dna polymerase must
accurately do its function this refers
to the ability of the polymerase to
avoid
or to correct errors in the newly
synthesized
dna strand thus this polymerase
proofreads the newly synthesized dna
and the polymerase usually this is dna
polymerase 1
removes the incorrect nucleotides
immediately after they are added to the
growing
dna during replication
the dna polymerase commits an error
in hydrogen bonding
leading to mispairing of the bases
meaning the base pairs may not be
complementary
and this happens once in every 10 to the
fourth
to 10 to the fifth base pairs what is
the meaning of that
if you have for example a
about 10 to the nine base pairs in the
chromosome
the error occurs every 10 to the fourth
base the next one would be
10 to the 8th so in this particular
example there are two errors that occur
but these errors are usually corrected
by
the proofreading
done by dna polymerase because of that
finally errors
occur only in every 10 to the 9 to 10 to
the 10
base pairs that is for in this example
if you have 10 to the nine base pairs of
uh
your dna there would only be one error
but many bnas
are less than 10 to the nine so you
might there might be no mutation at all
so proofreading would
decrease the frequency of
this error committed by
the dna polymerase
okay what is mutation it is a change in
the base sequence of
the dna any change in the base sequence
of the dna
and mutation may occur in two ways one
is by
spontaneous mutation
and the other is induced mutation
so i have mentioned already spontaneous
mutation
in the previous slide it occurs during
normal genetic function meaning it
occurs
normally during the activity of the dna
polymerase when it
synthesizes the dna
and what happens there is that
the dna polymerase may cause
error by misspearing the bases as i have
already mentioned
spontaneous mutation may also occur
through spontaneous uh hydrolytic
reactions
so there are there may be some
hydrolytic reactions within the cell
that would cause the modification of
the base so for example if a base has an
amino group
a hydrolytic reaction may remove the
amino group of that base
and that happens that base may
erroneously pair
with a different partner
so that again causes base mispairing
and we already mentioned that
spontaneous mutation
may occur every
10 to the 9 10 to 10 to the 10
base pairs or
the frequency is described as 10 to the
minus 9 to 10 to the minus 10 per
generation
this frequency may be increased
by induced mutation
when it is induced by mutagens
the frequency may become 10 to the minus
3
to 10 to the minus 4 pair generation so
it looks like
um the frequency becomes something like
the error committed by dna polymerase
when there is no proof reading but
remember that this is induced by
mutagens so it may be increased to
10 to the 3 10 to the minus 3 to 10 to
the minus 4 per generation meaning that
if you have 10 to the nine base pairs
there may be there may be
two to three sides
of changes
the dna so it is more frequent than
in spontaneous mutation
and with the kind
with the lifestyle that we have now and
the environment that we have
we are exposed to many mutagens
that would increase the frequency of
mutation
what are these mutagens they may include
physical agents
particularly radiation
radiation include ultraviolet light
and ionizing radiation
ionizing radiation includes x-rays
gamma rays and then chemicals there are
also chemicals that can cause
changes in the dna examples are
nitrous acid intercalating agents
fibromyalgia and
the so-called base analogues
let's see what ultraviolet light does to
our dna
we are constantly exposed to ultraviolet
light
and because of our
melanin we might be
protected from ultraviolet light
effects but white people
who have less melanin may not be
protected this is what happens when
ultraviolet light
strikes our dna
so this is the normal structure of the
dna you have here the sugar phosphate
backbone
where the bases are attached
ultraviolet light usually affects
the part of the dna where there are two
adjacent
pyrimidines this pyrimidines
thymine here and another timing here
normally
have no covalent bond between them
the covalent bond is formed between the
sugar and
the base but if ultraviolet light
hits the dna it causes the formation of
covalent bond
between this bases
and a cyclobutane ring is formed
when this cyclobutane ring is formed
this distorts
the dna as you can see here it distorts
the conformation of the dna and this
will cause error in
replication
other rotation include
ionizing radiation so for example
x-ray when x-ray hits
our cells
it strikes
water which is uh of course
uh abundant in our cells
water is uh ionized
into hydrogen and hydroxyl
radicals so this uh this would ionize
water into hydroxyl
and hydrogen radicals
so that that's there
denotes radical formation and one
important characteristic of radicals is
that
they are very reactive they can easily
react to other molecules
like for example hydroxyl radical
it easily reacts with hydrogen which is
abundant in the dna
it's very reactive such that it easily
forms water
and that the radical transfers to the
dna
it's now the dna that is a radical
now it's the dna that would be very
reactive
and in a chain of reactions eventually
the dna breaks
the break usually involves one strand a
single stranded braid
and breaks in the dna will also cause
error in replication and thus
mutation
chemical agents
so there are chemical agents that are
that have conformations that are similar
to the bases
to the pyrimidines and the purines
these are called base analogues so be
careful when you are
handling this kind of
base analogues or chemicals these
analogs
resemble the usual bases in our dna
and they may incorporate in the dna
as in this example you have five bromo
uracil
here fibromyalgia it looks like uracil
except that there is
a bromine group here
so that is not a natural base in our dna
it can in it can insert
in the dna it can be integrated here in
the dna
so that when this dna replicates
this would be read by the dna polymerase
as a u and
thus an a would be inserted in the new
dna a new base using or nucleotide is
inserted in the
dna and that results in
mutation
other chemicals include so-called
intercalating
agents intercalating agents
are planar compounds that are able to
insert
between the stock bases
this causes distortion of the global
helix
once again such distortion would cause
error
in replication
example of intercalating agents
are you have bencipirin
benzopyrene which is found in
burned meat no when you eat barbecue
and the meat is burned that's
rich in benzopyrene and benzopyrene
would intercalate with the
dna that's why bbq may be carcinogenic
there are also reagents that we use in
the laboratory that are intercalating
agents like for example
um
what do you call this uh ethidium
bromide which we use to
to visualize dna
and um acridine dyes
these are intercalating agents