Protein Synthesis

(Lecture # 5)
Protein Synthesis
• Protein synthesis is called translation because information
present as a nucleic acid sequence is translated into a different
language, the sequence of amino acids in a protein.
• This complex process is mediated by the coordinated interplay
of more than a hundred macromolecules, including
• mRNA
• rRNAs
• tRNAs
• Aminoacyl-tRNA synthetases
• Protein factors
• Any alteration in the nucleic acid sequence may result in an
incorrect amino acid being inserted into the polypeptide chain,
potentially causing disease or even death of the organism.
Components Required for Translation
1. Amino acids
• All the amino acids that eventually appear in the finished protein must be
present at the time of protein synthesis. If one amino acid is missing,
translation stops at the codon specifying that amino acid.
2. Transfer RNA
• In humans, there are at least 50 species of tRNA, whereas bacteria contain at
least 30 species
• Amino acid attachment site
• —CCA sequence at the 3'-end of the tRNA
• Anticodon:
• a three-base nucleotide sequence, the anticodon, that pairs with a specific codon on the
mRNA
3. Aminoacyl-tRNA synthetases:
• Family of enzymes required for
attachment of amino acids to their
corresponding tRNAs
• In addition to their synthetic activity, the
aminoacyl-tRNA synthetases have a
“proofreading” or “editing” activity that
can remove an incorrect amino acid
from the enzyme or the tRNA molecule.
4. Messenger RNA
• Template for the synthesis of the desired polypeptide chain must be present
5. Functionally competent ribosomes
• The small ribosomal 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 in the tRNA.
• The large ribosomal subunit catalyzes formation of
the peptide bonds that link amino acid residues in a
protein.
• The RER-associated ribosomes synthesize proteins
• exported from the cell
• incorporated into plasma
• endoplasmic reticulum, or Golgi membranes or
imported into lysosomes
• Cytosolic ribosomes synthesize proteins for
• cytosol
• nucleus
• mitochondria or peroxisomes
6. Protein factors
• Initiation, elongation, and termination (or release ) factors are required for
peptide synthesis.
• Some of these protein factors perform a catalytic function, whereas others
appear to stabilize the synthetic machinery
7. ATP and GTP are required as sources of energy
 Wobble Hypothesis
• The mechanism by which tRNAs can recognize more
than one codon for a specific amino acid is
described by the “wobble” hypothesis, which states
that codon–anticodon pairing follows the traditional
Watson-Crick rules (C pairs with G and A pairs with
U) for the first two bases of the codon but can be
less stringent for the last base.
Steps in Protein Synthesis
• The process of protein synthesis translates the 3-letter alphabet of nucleotide
sequences on mRNA into the 20-letter alphabet of amino acids that constitute proteins.
• The mRNA is translated from its 5'-end to its 3'-end, producing a protein synthesized
from its amino (N)-terminal end to its carboxyl (C)-terminal end.
• Prokaryotic mRNAs often have several coding regions (that is, they are polycistronic.
Each coding region has its own initiation and termination codon and produces a
separate species of polypeptide.
• In contrast, each eukaryotic mRNA has only one coding region (that is, it is
monocistronic).
• The process of translation is divided into three separate steps:
• initiation
• elongation
• termination
A. Initiation
• Initiation of protein synthesis involves the assembly of the
components of the translation system before peptide bond formation
occurs .
• These components include:
• ribosomal subunits
• mRNA to be translated
• aminoacyl-tRNA
• GTP
• Prokaryotic initiation factors (IF-1, IF-2, and IF-3), whereas in
• Eukaryotic initiation factors, (eIF)
The following are two mechanisms by which the ribosome recognizes the nucleotide sequence (AUG) that initiates translation.

1. Shine-Dalgarno sequence:
• In Escherichia coli (E. coli), a purine rich sequence of nucleotide bases, 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 rRNA component of the small (30S) ribosomal subunit has a nucleotide sequence near its 3'-end
that is complementary to all or part of the SD sequence.
• Therefore, the 5'-end of the mRNA and the 3'-end of the 16S rRNA can form complementary base pairs,
facilitating the positioning of the small ribosomal subunit on the mRNA in close proximity to the initiating
AUG codon.

2. 5' Cap:
• Eukaryotic mRNAs do not have SD sequences.
• In eukaryotes, the small (40S) ribosomal subunit (aided by members of the elF-4 family of proteins) binds
close 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.
3. Initiation codon:

• The initiating AUG is recognized by a special initiator tRNA.

• Recognition is facilitated by IF-2-GTP in prokaryotes and
eIF-2-GTP (plus additional eIFs ) in Eukaryotes.
• In Prokaryotes, initiator tRNA carries N-formylated methionine
(fMet)
• In Eukaryotes, initiator tRNA carries methionine
B. Elongation

• Elongation of the polypeptide chain involves the addition of amino acids to the carboxyl end of the growing chain.

• During elongation, the ribosome moves from the 5'-end to the 3'-end of the mRNA that is being translated.
elongation Factors EF-Tu-GTP and EF-Ts and requires GTP hydrolysis.

• The formation of the peptide bond is catalyzed by peptidyl transferase, an activity intrinsic to the 23S rRNA found in
the large (50S) ribosomal subunit.

• After the peptide bond has been formed, what was attached to the tRNA at the P site is now linked to the amino acid
on the tRNA at the A site. The ribosome then advances three nucleotides toward the 3'-end of the mRNA. This
process is known as translocation and, in prokaryotes, requires the participation of EF-G-GTP (eukaryotic cells use EF-
2-GTP) and GTP hydrolysis.

• Translocation causes movement of the uncharged tRNA from the P to the E site for release and movement of the
peptidyltRNA from the A to the P site. The process is repeated until a termination codon is encountered.
Formation of a peptide bond.
• Peptide bond formation involves transfer of the
peptide on the transfer RNA (tRNA) in the P site to the
amino acid on the tRNA in the A site (transpeptidation)
C. Termination

• Termination occurs when one of the three termination codons moves into the A site.

• These codons are recognized in E. coli by release factors: RF-1, which recognizes the termination codons UAA and
UAG, and RF-2, which recognizes UGA and UAA.

• The binding of these release factors results in hydrolysis of the bond linking the peptide to the tRNA at the P site,
causing the nascent protein to be released from the ribosome. A third release factor, RF-3-GTP then causes the
release of RF-1 or RF-2 as GTP is hydrolyzed.

• Eukaryotes have a single release factor, eRF, which recognizes all three termination codons.
Protein factors in the three stages of translation
Polysomes
• Translation begins at the 5'-end of the mRNA, with the ribosome proceeding
along the RNA molecule. Because of the length of most mRNAs, more than one
ribosome at a time can translate a message. Such a complex of one mRNA and a
number of ribosomes is called a polysome or polyribosome.

Fig. Polyribosome
Regulation of Translation
• Gene expression is most commonly regulated at the transcriptional
level, but translation may also be regulated.
• An important mechanism by which this is achieved in eukaryotes is by
covalent modification of eIF-2: phosphorylated eIF-2 is inactive.
• In both eukaryotes and prokaryotes, regulation can also be achieved
through proteins that bind mRNA and inhibit its use by blocking
translation or extend its use by protecting it from degradation.
CO- AND POSTTRANSLATIONAL MODIFICATION
OF POLYPEPTIDE CHAINS
• Many polypeptide chains are covalently modified, either while they
are still attached to the ribosome (cotranslational) or after their
synthesis has been completed (posttranslational).
• 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. Examples are:
• Trimming
• Covalent attachments
• Phosphorylation
• Glycosylation
• Hydroxylation
Trimming
• Phosphorylation occurs on the hydroxyl groups of serine ;
threonine ; or, less frequently, tyrosine residues in a protein.
• This phosphorylation is catalyzed by one of a family of protein
kinases and may be reversed by the action of cellular protein
phosphatases.
• The phosphorylation may increase or decrease the functional
activity of the protein.
Proline and lysine residues of the alpha chains of
collagen are extensively hydroxylated by vitamin C-
dependent hydroxylases in the endoplasmic reticulum.
• Many of the proteins that are destined to become part of a
plasma membrane or to be secreted from a cell have
carbohydrate chains added enbloc to the amide nitrogen of
asparagine (N-linked) or built sequentially on the hydroxyl
groups of serine, threonine, or hydroxylysine (O-linked).
• N-glycosylation occurs in the endoplasmic reticulum and O-
glycosyation in the Golgi.
• Glycosylation is also used to target proteins to the matrix of
lysosomes.
Protein Degradation

• Proteins that are defective (for example,

misfolded) or destined for rapid turnover
are often marked for destruction by
ubiquitination, the attachment of chains
of a small, highly conserved protein,
called ubiquitin.
• Proteins marked in this way are rapidly
degraded by a cellular component known
as the proteasome, which is a
macromolecular, ATP-dependent,
proteolytic system located in the cytosol.