• Nucleus-Cytoplasmic RNA Transport

• Unlike bacteria, the eukaryotic cell is

compartmentalized. Thus, the primary
transcript is produced in one compartment
(the nucleus) but it must function in another
compartment (the cytoplasm).

• Therefore, the final processed product

(mRNA) must be transported through the
nuclear envelope to reach the cytoplasm and
be engaged with the ribosomes for
translation.
• There are specific pathways for the transport
of the various RNAs and that this involves
recognition of the RNA by transport activities
that allow the RNA to exit through the
complex nuclear pore structure.
Translation/ Protein synthesis
• Translation/ Protein synthesis
• Is called translation because the “language” of the
nucleotide sequence/ genetic message on the mRNA is
translated into the language of an amino acid sequence.

• The process of translation requires a genetic code.

 Genetic code
• Amino acid sequence of a polypeptide chain is
determined by the nucleotide sequence of mRNA.

• The relationship is determined by a genetic code,

dictionary of genetic words or codons.

• The genetic information contained in mRNA is read

in groups of three nucleotides (triplet of nucleotide
bases) called genetic words or codons.
 Characteristics Genetic Code
(i) A collection of 64 codons make up the genetic code.
• 61 codons code for 20 amino acids
• 3 codons are terminal / stop codons
• Two or more codons that specify for the same AA are
called synonyms
• Methionine and tryptophan are coded by single codons
• UAA, UGA, UAG, do not specify any AA and act as
termination/ stop/ non-sense codons.
• AUG- methionine or initiation/ start codon.
(ii) Degeneracy (redundancy)
• A given AA may be coded by more than one codon.

(iii) Universality
• The genetic code (with a few exception) is universal in
all living organisms (exception occurs in mitochondria)

(iv) Specificity/ Unambiguity

• Two or more different AA are not coded by the same
codon
(v) Non-overlapping and commaless
• The code is read from a fixed starting point as a
continuous sequence of bases, taken three at a time.
For example, AUCGUAACUACCAUA is read as AUC-
GUA-ACU-ACC-AUA without any “punctuation”
between the codons.

(iv) Colinear
• During Translation, mRNA is read 5’-3’ direction and
the protein is synthesized from N-terminal to C-
terminal direction
Steps in Protein Synthesis
• Protein synthesis occurs in ribosomes.

• The mRNA is translated from its 5′-end to 3′-end,

producing a protein synthesized from its amino-
terminal end to its carboxyl-terminal end.

• The process of translation is divided into three

separate steps: initiation, elongation, and termination.

• The polypeptide chains produced may be modified by

posttranslational modification.
1. Initiation

• Initiation of protein synthesis involves the assembly of

the components of the translation system before
peptide bond formation occurs.

• These components include the two ribosomal

subunits, the mRNA to be translated, the aminoacyl-
tRNA specified by the first codon in the message, GTP
(which provides energy for the process), and initiation
factors that facilitate the assembly of the initiation
complex
 Initiation factors
• In prokaryotes, three initiation factors are known (IF-1,
IF-2, and IF-3)
• In eukaryotes, there are over ten (designated eIF to
indicate eukaryotic origin)
(a) Shine-Dalgarno sequence:
• In Escherichia coli, a purine-rich sequence of
nucleotide bases (for example, 5′-UAAGGAGG-3′),
known as the Shine-Dalgarno (SD) sequence, is
located six to ten bases upstream of the initiating
AUG codon on the mRNA molecule—that is, near its
5′-end.
• The 16S ribosomal RNA component of the 30S
ribosomal subunit has a nucleotide sequence near its
3′-end that is complementary to all or part of the SD
sequence.
• The mRNA 5′-end and the 3′-end of the 16S ribosomal
RNA can form complementary base pairs
• Facilitating the binding and positioning of the 30S
ribosomal subunit on the mRNA in close proximity to
the initiating AUG codon
• In eukaryotes, the 40S ribosomal subunit (aided by
members of the elF-4 family of proteins) binds to the
cap structure at the 5′-end of the mRNA and moves
down the mRNA until it encounters the initiator AUG.
This “scanning” process requires ATP.
(b) Initiation codon:
• The initiating AUG is recognized by a special initiator
tRNA. Recognition is facilitated by IF-2 (bound to
GTP) in prokaryotes and eIF2-GTP (plus additional
eIFs) in eukaryotes
• Ribosome has three
binding sites for tRNA
molecules
• A – site; binds an
incoming aminoacyl-
tRNA
• P – site; the tRNA
chemically linked to the
growing polypeptide
chain is located here.
• E - site is occupied by
the empty tRNA as it is
about to exit the
ribosome.
1. Initiation of translation in eukaryotes
• The amino acid–charged initiator,Met-tRNA, enters
the ribosomal P site, and GTP is hydrolyzed to GDP.
[Note: The initiator tRNA is the only tRNA recognized
by eIF-2, and the only tRNA to go directly to the P
site.] (Met- methionine)
• The eIF4 bound cap of the mRNA binds to the
preinitiation complex to form initiation complex
• The initiation complex scan the mRNA to find AUG
and positioning of the small subunit and Met-tRNA at
the start codon (AUG).
Wobble Pairing of the tRNA anticodon with the
mRNA codon proceeds from the 5' end of the codon.
Once the first two positions are paired, exact base
pairing of the third position is less critical.
NOTE:
• The small subunit binds mRNA and is responsible
for the accuracy of translation by ensuring correct
base-pairing between the codon in the mRNA and
the anticodon of the tRNA.

• In bacteria and in mitochondria, the initiator tRNA

carries an N-formylated methionine
• Association of the large subunit (60S) forms as 80S
ribosome ready to translate mRNA
2. Elongation

• Elongation of the polypeptide chain involves the

addition of amino acids to the carboxyl end of the
growing chain.
• Once the 80S ribosome with Met-tRNA in the
ribosome P site is assembled, the second amino acid
(AA) coded by the mRNA binds to the A site. EFs are
also involved.
• The large rRNA subunit catalyzes peptide bond
formation between Met and new AA
• Ribosomal RNA component of the large subunit that
carries the enzymatic activity of peptidyl transferase.
• Hydrolysis of GTP in EF2.GTP causes conformational
change in the ribosome that results in its translocation
one codon along the mRNA
• Shifts the unacylated tRNA (uncharged) for Met to the
E site and the tRNA with the bound peptide to the P
site
• The cycle can begin with binding of tRNA bearing AA
to the open A site
3. Termination
• When the ribosomes bearing a nascent protein chain
reaches a stop codon (UAA, UGA, UAG), Release
Factor, eRF1, enters the ribosomal complex, probably
at or near the A site together with the eRF3.GTP.
• Hydrolysis of the bound GTP is accompained by
cleavage of the peptide chain from the tRNA in the P
site and release of the tRNAs an the two ribosomal
units
 Polysomes
• Multiple ribosomes can simultaneously translate a
eukaryotic mRNA
• When a ribosome completes translation and dissociates
from 3’ end, the separated subunits can rapidly find the
nearby 5’ cap and initiate another round of synthesis
Posttranslational Modification of Polypeptide Chains

• The modifications occur after translation is initiated,

they are called posttranslational modifications.
• These modifications may include removal of part of
the translated sequence,
• Or the covalent addition of one or more chemical
groups required for protein activity
A. Trimming
Many proteins destined for secretion from the cell are
initially made as large, precursor molecules that are
not functionally active.
Portions of the protein chain must be removed by
specialized endoproteases, resulting in the release of
an active molecule.
Some precursor proteins are cleaved in the
endoplasmic reticulum or the Golgi apparatus, others
are cleaved in developing secretory vesicles (for
example, insulin and some precursor proteins, such
as collagen are cleaved after secretion).
Insulin

Collagen
Zymogens are inactive precursors of secreted
enzymes (including the proteases required for
digestion).
They become activated through cleavage when they
reach their proper sites of action. For example, the
pancreatic zymogen, trypsinogen, becomes activated
to trypsin in the small intestine.
B. Covalent alterations
1. Phosphorylation:
Phosphorylation occurs on the hydroxyl groups of
serine, threonine, or tyrosine residues in a protein.
Phosphorylation is catalyzed by one of a family of
protein kinases
The phosphorylation may increase or decrease the
functional activity of the protein.
2. Glycosylation:

The addition of sugars occurs in the endoplasmic

reticulum and the Golgi apparatus.

3. Hydroxylation:
Proline and lysine residues of the α chains of collagen
are extensively hydroxylated in the endoplasmic
reticulum
4. Other covalent modifications:

These may be required for the functional activity of a

protein.
For example, additional carboxyl groups can be
added to glutamate residues by vitamin K–dependent
carboxylation.
The resulting γ-carboxyglutamate residues are
essential for the activity of the several blood-clotting
factors/ proteins.
Mutations
1. Changing a single nucleotide base on the mRNA
chain (a “point mutation”) can lead to any one of three
results
(i) Silent mutation
(ii) Missense mutation
(iii) Nonsense mutation
(i) Silent mutation:
• The codon containing the changed base may code for
the same amino acid.
• Serine codon UCA. If a mutation occurs at the third
base to become UCU
(ii) Missense mutation:
• The codon containing the changed base may code for
a different amino acid
• Serine codon UCA. If a mutation occurs at the first
base to become CCA
(iii) Nonsense mutation :
• The codon containing the changed base may become
a termination codon
• Serine codon UCA. If a mutation occurs at the second
base to become UAA
(2) Other mutation : These can alter the amount or
structure of the protein produced by translation.

(a) Trinucleotide repeat expansion:

• A sequence of three bases that is repeated in tandem
(one behind another) will become amplified in number,
so that too many copies of the triplet occur.
• If this occurs within the coding region of a gene, the
protein will contain many extra copies of one amino
acid.
(b) Splice site mutations:
• Mutations at splice sites can alter the way in which
introns are removed from pre-mRNA molecules
(c) Frame-shift mutations:
• If one or two nucleotides are either deleted from or
added to the coding region of a message sequence, a
frame-shift mutation occurs and the reading frame is
altered.
• If three nucleotides are added, a new amino acid is
added to the peptide or, if three nucleotides are
deleted, an amino acid is lost.
• In these instances, the reading frame is not affected.
• Loss of three nucleotides maintains the reading
frame, but can result in serious pathology.
• For example, cystic fibrosis (CF), a hereditary
disease that primarily affects the pulmonary and
digestive systems