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Lec.3 Protein synthesis
Genetic informations are stored in the chromosomes and
transmitted to daughter cells through DNA replication they are
expressed through transcription to RNA and, in the case of
mRNA subsequent translation into polypeptide chains, The
process of translation requires a genetic code, through which
the information contained in the nucleic acid sequence is
expressed to produce a specific sequence of amino acids. Any
alteration in the nucleic acid sequence may result in an
improper amino acid being inserted into the polypeptide chain,
potentially causing disease or even death of the organism.
Many polypeptide chains are covalently modified following
their synthesis to activate them,or alter their activities, or
target them to their final intracellular or extracellular
destinations.
The genetic code
Each genetic code is composed of three nucleotide bases and
called codons.
Codons
Codons are usually presented in the mRNA as adenine (A),
guanine (G), cytosine (C), and uracil (U). Their nucleotide
sequences are always written from the 5'-end to the 3'-end.
The four nucleotide bases are used to produce the three-base
codons. There are, therefore, 64 different combinations of
bases, taken three at a time
Termination ("stop" or "nonsense") codons: three of the
codons, UAG, UGA, and UAA, do not code for amino acids,
but rather are termination codons. When one of these codons
appears in an mRNA sequence, it signals that synthesis of the
peptide chain coded for by that’s mRNA is completed
Components required for translation
A large number of components are required for the synthesis
of a polypeptide chain. These include
All the amino acids that are found in the finished product.
The mRNA to be translated.
tRNA.
functional ribosome
Energy sources.
Enzymes.
Protein factors needed for initiation, elongation, and
termination of the polypeptide chain.
A. Amino acids
All the amino acids that eventually appear in the finished
protein must be present at the time of protein synthesis. [Note:
if one amino acid is missing (for example, if the diet does not
contain an essential amino acid) that amino acid is in limited
supply in the cell, and translation therefore, stops at the
codon specifying that amino acid. This demonstrates the
importance of having all the essential amino acids in sufficient
quantities in the diet to ensure continued protein synthesis
B. Transfer RNA (tRNA)
Each tRNA molecule has an attachment site for a specific
amino acid and an Anticodon (which is a three-base
nucleotide sequence that recognizes a specific codon on the
mRNA this codon specifies the insertion into the growing
peptide chain of the amino acid carried by that tRNA, The
amino acid that is attached to the tRNA molecule is said to be
activated
C. Aminoacyl -tRNA synthetases
This enzyme is required for attachment of amino acids to their
corresponding tRNAs. Each member of this family recognizes
a specific amino acid and the tRNAs that correspond to that
aminoacid. The extreme specificity of this enzyme in
recognizing both the amino acid and and its specific tRNA is
largely responsible for the high fidelity of translation of the
genetic message in addition this enzyme have a "proofreading"
activity that can remove amino acids from the tRNA molecule.
D. Messenger RNA:
The specific mRNA required as a template for the synthesis of
the desired polypeptide chain must be present
E. Ribosomes:
Ribosomes are large complexes of protein and rRNA,
ribosomes serve as "factories" in which the synthesis of
proteins occurs,It is composed of
Ribosomal RNA (rRNA)
Ribosomal proteins
3 Binding sites on the ribosomes called A, P,& E for tRNA
they cover three neighboring codons. During translation, the
A site binds an incoming (aminoacid –tRNA) as directed by
the codon currently occupying this site.The P site codon is
occupied by peptidyl –tRNA This tRNA carries the chain of
amino acids that has already been synthesized. The E site is
occupied by the empty tRNA as it is about to exit the ribosom
F. Protein factors: they perform a catalytic function, or stabilize
the synthetic machinery.
G. Sources of energy: ATP & GTP are required as energy source
CODON RECOGNITION BY tRNA:
Recognition of a particular codon in an mRNA sequence is
accomplished by the anticodon sequence of the tRNA, Some
tRNAs recognize more than one codon for a given amino acid
Antiparallel binding between codon and anticodon
Binding of the tRNA anticodon to the mRNA codon follows
the rules of complementary and antiparallel binding, that is,
the mRNA codon is "read" 5-3 by an anticodon pairing in the
"flipped” 3-5 orientation
Steps in protein synthesis:
The pathway of protein synthesis translates the nucleotide
sequences on the mRNA into amino acids that constitute
proteins. The process of translation is divided into three
separate steps: initiation, elongation, and termination. The
polypeptide chains produced may be modified by
posttranslational modification.
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A. 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, specified by
the first codon in the message, GTP (which provides energy
for the process), and initiation factors that facilitate the
assembly of this initiation complex
The main mechanism by which translation is initiated :
Ribosomal subunit binds to the cap structure at the 5'-end
of the mRNA and moves down the mRNA until it
encounter the initiator AUG codon.
Initiation codon: The initiating codon AUG is recognized
by a special initiator tRNA that enters the ribosomal P-site.
Note: Only the initiator tRNA goes to the P site- other
charged tRNAs enter at the A site
.
B. Elongation: Elongation of the polypeptide chain involves the
addition of amino acids to the carboxyl end of the growing
chain. facilitated. by elongation factors known as eEF The
formation of the peptide bonds is catalyzed by
peptidyltransferase . After the peptide bond has been formed,
the ribosome advances three nucleotides toward the 3'-end of
the mRNA. This processs known as translocation This causes
movement of the uncharged tRNA into the ribosomal E site
and movement of the peptidyl-tRNA into the P-site.
C. Termination:
Termination occurs when one of the three termination codons
(UAA, UAG, UGA) moves into the A site and by help of
release factors (RF ).These factors cause the newly
synthesized protein to be released from the ribosomal
complex, and, at the same time, cause the dissociation of the
ribosome from the mRNA.
D. D. Polysomes are complex of one mRNA and a number of
ribosomes are called a polysome or polyribosome.
E. Regulation of translation
Although gene expression is most commonly regulated at the
transcriptional level, the rate of protein synthesis is also
sometimes regulated. The translation of some messenger RNA
molecules is regulated by the binding of regulatory proteins,
which sometimes block translation, and sometimes stabilize
the mRNA to extend its life time.
Posttranslational modification of polypeptide
chains
Many polypeptide chains are modified, either while they are
still attached to the ribosome or after their synthesis has been
completed. Because the modifications occur after translation is
initiated they are called posttranslational modifications.. Some
types of posttranslational modifications are listed below.
A. Trimming
Many proteins are initially made as large, precursor molecules
that are not functionally active Portions of the protein chain
must be removed by specialized endo proteases, resulting in
the release of an active molecule.
B. Covalent alterations
Protein both enzymatic and structural may be activated or
inactivated by the covalent attachment of a variety of chemical
groups Examples of these modifications include.
Phosphorylation,.glycosylation, hydroxylation
C. Protein degradation
Proteins that are defective are often marked for destruction by
ubiquitination which is the attachment of a small protein,
called ubiquitin and is rapidly degraded by a cellular
component known as the "proteasome"
Consequences of altering the nucleotide sequence:
Changing a single nucleotide base on the mRNA chain (a
"pointmutation") can lead to any one of three results
1) Silent mutation: The codon containing the changed base may
code for the same amino acid. For example, if the serine codon
UCA is given a different third base U to become CCA it still
codes for serine. Therefore, this is termed a "silent" mutation.
2) Missense mutation: The codon containing the changed base
maycode for a different amino acid. For example, if the serine
codonUCA is given a different first base -C become CCA, it
will code for a different amino acid, in this case, proline. This
substitution of an incorrect amino acid is called a "missense"
mutation.
3) Nonsense mutation: The codon containing the changed base
may become a termination codon. For example, if the serine
codon UCA is given a different second base-A- become UAA,
the new codon causes termination of translation at that point.
Thecreation of a termination codon at an inappropriate place is
called a "nonsense" mutation.
4) Other mutations: These can alter the amount or structure of
theprotein produced by translation.
a) Trinucleotide repeat expansion: Occasionally, a
sequence of three bases that is repeated in tandem 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. For example, amplification of the CAG codon
leads to the insertion of many extra glutamine residues in
the huntingtin protein, causing the huntingtin disease.
b) Splice site mutations: Mutations at splice sites can alter
the way in which introns are removed from pre mRNA
molecules, producing aberrant proteins.
c) Frame-shift mutations: if one or two nucleotides are
either deleted from or added to the interior of a message
sequence, a frame-shift mutation occurs and the reading
frame is altered. The resulting amino acid sequence may
become radically different from this point on. [Note: if 3
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