TRANSLATION

BY DR. BUSHRA HAMED

MIMS.
• The conventional concept of central dogma of life which in essence is
“DNA makes RNA makes protein” is an oversimplification of molecular
biology.
• With the advances in cell biology and rapid developments in
bioinformatics, the terms genome, transcriptome and proteome are in
current use to represent the central dogma of life.
• GENOME: The total DNA (genetic information) contained in an
organism or a cell is regarded as the genome.
• Thus, the genome is the storehouse of biological information. It
includes the chromosomes in the nucleus and the DNA in
mitochondria, and chloroplasts.
• GENOMICS: The study of the structure and function of genome is
genomics.
• The term functional genomics is used to represent the gene
expression and relationship of genes with gene products.
• Structural genomics refers to the structural motifs and complete
protein structures.
• Comparative genomics involves the study of comparative gene
function and phylogeny.
• Metagenomics refers to the study of genomes of whole
communities of microscopic life.
• TRANSCRIPTOME: The RNA copies of the active protein coding genes
represent transcriptome.
• Thus, transcriptome is the initial product of gene expression which
directs the synthesis of proteins.
• TRANSCRIPTOMICS: The study of transcriptome that involves all the
RNA molecules made by a cell, tissue or an organism is transcriptomics.
• PROTEOME: The cell’s repertoire (repository/storehouse) of proteins
with their nature and biological functions is regarded as proteome.
• Thus, proteome represents the entire range of proteins and their
biological functions in a cell.
• PROTEOMICS: The study of the proteome.
• METABOLOMICS: The use of genome sequence analysis for
determining the capability of a cell, tissue or an organism to
synthesise small molecules (metabolites) is metabolomics.
• TRANSLATION: The genetic information stored in DNA is
passed on to RNA (through transcription), and ultimately
expressed in the language of proteins.
• The biosynthesis of a protein or a polypeptide in a living cell is
referred to as translation.
• The term translation is used to represent the biochemical
translation of four-letter language information from nucleic
acids (DNA and then RNA) to 20 letter language of proteins.
• The sequence of amino acids in the protein synthesised is
determined by the nucleotide base sequence of mRNA.
• VARIABILITY OF CELLS IN TRANSLATION: There are wide variations in the
cells with respect to the quality and quantity of proteins synthesised.
• This largely depends on the need and ability of the cells.
• Erythrocytes (red blood cells) lack the machinery for translation, and
therefore cannot synthesise proteins.
• In general, the growing and dividing cells produce larger quantities of
proteins.
• Some of the cells continuously synthesise proteins for export.
• For instance, liver cells produce albumin and blood clotting factors for
export into the blood for circulation.
• The normal liver cells are very rich in the protein biosynthetic machinery,
and thus the liver may be regarded as the protein factory in the human
body.
• GENETIC CODE: The three nucleotide (triplet) base sequences in mRNA
that act as code words for amino acids in protein constitute the genetic
code or simply codons.
• The genetic code is regarded as a dictionary of nucleotide bases (A, G, C
and U) that determines the sequence of amino acids in proteins.
• The codons consist of the four nucleotide bases, the purines - adenine (A)
and guanine (G), and the pyrimidines - cytosine (C) and uracil (U).
• These four bases produce 64 different combinations of three base codons.
• The nucleotide sequence of the codon on mRNA is written from
the 5’ end to 3’ end.
• Sixty one codons code for the 20 amino acids found in protein.
• The three codons UAA, UAG and UGA do not code for amino
acids. They act as stop signals in protein synthesis.
• These three codons are collectively known as termination
codons or non-sense codons.
• The codons UAG, UAA and UGA are often referred to,
respectively, as amber, ochre and opal codons.
• The codons AUG - and, sometimes, GUG - are the chain initiating
codons.
• OTHER CHARACTERISTICS OF GENETIC CODE: The genetic code is
universal, specific, non-overlapping and degenerate.
• UNIVERSALITY: The same codons are used to code for the same
amino acids in all the living organisms. Thus, the genetic code has
been conserved during the course of evolution. Hence genetic code is
appropriately regarded as universal.
• There are, however, a few exceptions. For instance, AUA is the codon
for methionine in mitochondria. The same codon (AUA) codes for
isoleucine in cytoplasm.
• SPECIFICITY: A particular codon always codes for the same amino
acid, hence the genetic code is highly specific or unambiguous, ex:
UGG is the codon for tryptophan.
• NON-OVERLAPPING: The genetic code is read from a fixed point as a
continuous base sequence.
• It is non-overlapping, commaless and without any punctuations. For
instance, UUUCUUAGAGGG is read as UUU/CUU/AGA/GGG.
• Addition or deletion of one or two bases will radically change the
message sequence in mRNA.
• And the protein synthesised from such mRNA will be totally different.
• This is encountered in frameshift mutations which cause an
alteration in the reading frame of mRNA.
• DEGENERATE: Most of the amino acids have more than one
codon.
• The codon is degenerate or redundant, since there are 61
codons available to code for only 20 amino acids.
• For instance, glycine has four codons. The codons that
designate the same amino acid are called synonyms. Most of
the synonyms differ only in the third (3’-end) base of the
codon.
• CODON-ANTICODON RECOGNITION: The codon of the mRNA is
recognised by the anticodon of tRNA.
• They pair with each other in antiparallel direction (5’- 3’) of mRNA with
(3’ - 5’) of tRNA.
• The usual conventional complementary base pairing (A=U, C=G)
occurs between the first two bases of codon and the last two bases of
anticodon.
• The third base of the codon is rather lenient or flexible with regard to
the complementary base.
• The anticodon region of tRNA consists of seven nucleotides and it
recognises the three letter codon in mRNA
• WOBBLE HYPOTHESIS: Wobble hypothesis, put forth by Crick,
is the phenomenon in which a single tRNA can recognise more
than one codon.
• This is due to the fact that the third base (3’- base) in the
codon often fails to recognise the specific complementary base
in the anticodon (5’- base).
• Wobbling is attributed to the difference in the spatial
arrangement of the 5’-end of the anticodon.
• The possible pairing of 5’-end base of anticodon (of tRNA) with
the 3’-end base of codon (mRNA) is given
• Wobble hypothesis explains the degeneracy of the genetic
code, i.e. existence of multiple codons for a single amino acid.
• Although there are 61codons for amino acids, the number of
tRNAs is far less (around 40) which is due to wobbling.
• CODON BIAS: Many amino acids have multiple codons.
However, the organisms prefer to use one or two codons (not
all of them), and this phenomenon is referred to as codon bias.
• It is variable, depending the organism. As a result of codon
bias, low amounts of tRNAs for the rarely used codons are
made.
COMPLEMENTARY BINDING OF CODON
OF mRNA AND ANTICODON OF tRNA
• MUTATIONS AND GENETIC CODE: Mutations result in the change
of nucleotide sequences in the DNA, and consequently in the RNA.
• The ultimate effect of mutations is on the translation through the
alterations in codons. Some of the mutations are harmful.
• The occurrence of the disease sickle cell anemia due to a single
base alteration (CTC - CAC in DNA, and GAG – GUG in RNA) is a
classical example of the seriousness of mutations.
• The result is that glutamate at the 6th position of β-chain of
haemoglobin is replaced by valine. This happens since the altered
codon GUG of mRNA codes for valine instead of glutamate.
• Frameshift mutations are caused by deletion or insertion of
nucleotides in the DNA that generate altered mRNAs.
• As the reading frame of mRNA is continuous, the codons are
read in continuation, and amino acids are added.
• This results in proteins that may contain several altered amino
acids, or sometimes the protein synthesis may be terminated
prematurely.
• BIOSYNTHESIS OF PROTEIN: The protein synthesis which
involves the translation of nucleotide base sequence of mRNA
into the language of amino acid sequence may be divided into
the following stages.
1. Requirement of the components.
II. Activation of amino acids.
III. Protein synthesis proper.
IV. Chaperones and protein folding.
V. Post-translational modifications.
1. REQUIREMENT OF THE COMPONENTS: The protein synthesis may
be considered as a biochemical factory operating on the ribosomes.
• As a factory is dependent on the supply of raw materials to give a
final product, the protein synthesis also requires many components.
• AMINO ACIDS: Proteins are polymers of amino acids.
• Of the 20 amino acids found in protein structure, half of them 10 can
be synthesised by man. About 10 essential amino acids have to be
provided through the diet.
• Protein synthesis can occur only when all the amino acids needed for
a particular protein are available.
• If there is a deficiency in the dietary supply of any one of the essential amino
acids, the translation stops.
• It is, therefore, necessary that a regular dietary supply of essential amino acids,
in sufficient quantities, is maintained, as it is a prerequisite for protein
synthesis.
• As regards prokaryotes, there is no requirement of amino acids, since all the 20
are synthesised from the inorganic components.
• RIBOSOMES: The functionally active ribosomes are the centres or factories for
protein synthesis.
• Ribosomes may also be considered as workbenches of translation.
• Ribosomes are huge complex structures (70S for prokaryotes and 80S for
eukaryotes) of proteins and ribosomal RNAs.
• Each ribosome consists of two subunits, one big and one small. The
functional ribosome has two sites, A site and P site.
• Each site covers both the subunits.
• A site is for binding of aminoacyl tRNA and P site is for binding peptidyl
tRNA, during the course of translation.
• In case of eukaryotes, there is another site called exit site or E site.
Thus, eukaryotes contain three sites (A, P and E) on the ribosomes.
• The ribosomes are located in the cytosomal fraction of the cell.
• They are found in association with rough endoplasmic reticulum (RER)
to form clusters RER- ribosomes, where the protein synthesis occurs.
• The term polyribosome (polysome) is used when several ribosomes
simultaneously translate on a single mRNA.
• mRNA: The specific information required for the synthesis of a given
protein is present on the mRNA.
• The DNA has passed on the genetic information in the form of codons to
mRNA to translate into a protein sequence.
• tRNA: They carry the amino acids, and hand them over to the growing
peptide chain. The amino acid is covalently bound to tRNA at the 3’-end.
• Each tRNA has a three nucleotide base sequence, the anticodon, which is
responsible to recognise the codon (complementary bases) of mRNA for
protein synthesis.
A POLYRIBOSOME IN PROTEIN SYNTHESIS
• In man, there are about 50 different tRNAs whereas in bacteria
around 40 tRNAs are found.
• Some amino acids (particularly those with multiple codons) have
more than one tRNA.
• ENERGY SOURCES: Both ATP and GTP are required for the supply of
energy in protein synthesis.
• Some of the reactions involve the breakdown of ATP or GTP,
respectively, to AMP and GMP with the liberation of pyrophosphate.
• Each one of these reactions consumes two high energy phosphates
(equivalent to 2 ATP).
• PROTEIN FACTORS: The process of translation involves a number of
protein factors.
• These are needed for initiation, elongation and termination of protein
synthesis. The protein factors are more complex in eukaryotes
compared to prokaryotes.
II. ACTIVATION OF AMINO ACIDS: Amino acids are activated and attached
to tRNAs in a two step reaction.
• A group of enzymes namely aminoacyl tRNA synthetases are required
for this process.
• These enzymes are highly specific for the amino acid and the
corresponding tRNA.
• The amino acid is first attached to the enzyme utilising ATP to
form enzyme-AMP-amino acid complex.
• The amino acid is then transferred to the 3’ end of the tRNA to
form aminoacyl-tRNA.
III. PROTEIN SYNTHESIS PROPER: The protein or polypeptide
synthesis occurs on the ribosomes (rather polyribosomes).
• The mRNA is read in the 5’-3’ direction and the polypeptide
synthesis proceeds from N-terminal end to C-terminal end.
• Translation is directional and collinear with mRNA.
FORMATION OF AMINOACYL-tRNA
• The prokaryotic mRNAs are polycistronic, since a single mRNA
has many coding regions that code for different polypeptides.
• In contrast, eukaryotic mRNA is monocistronic, since it codes
for a single polypeptide.
• In case of prokaryotes, translation commences before the
transcription of the gene is completed. Thus, simultaneous
transcription and translation are possible.
• This is not so in case of eukaryotic organisms since
transcription occurs in the nucleus whereas translation takes
place in the cytosol.
• Further, the primary transcript (hnRNA) formed from DNA has to
undergo several modifications to generate functional mRNA.
• INITIATION OF TRANSLATION: The initiation of translation in
eukaryotes is complex, involving at least ten eukaryotic initiation
factors (eIFs). Some of the eIFs contain multiple (3 - 8) subunits. The
process of translation initiation can be divided into four steps.
a) Ribosomal dissociation.
b) Formation of 43S preinitiation complex.
c) Formation of 48S initiation complex.
d) Formation of 80S initiation complex.
a) RIBOSOMAL DISSOCIATION: The 80S ribosome dissociates to
form 40S and 60S subunits.
• Two initiating factors namely eIF-3 and eIF-1A bind to the newly
formed 40S subunit, and thereby block its reassociation with 60S
subunit.
• For this reason, eIF-3 is known as anti-association factor.
b) FORMATION OF 43S PRE-INITIATION COMPLEX: A ternary
complex containing met-tRNAi and eIF-2 bound to GTP attaches to
40S ribosomal subunit to form 43S preinitiation complex.
• The presence of eIF-3 and eIF-1A stabilises this complex.
c) FORMATION OF 48S INITIATION COMPLEX: The binding of mRNA to 43S
preinitiation complex results in the formation of 48S initiation complex
through the intermediate 43S initiation complex.
• This, however, involves certain interactions between some of the eIFs
and activation of mRNA.
• eIF-4F complex is formed by the association of eIF-4G, eIF-4A with eIF-
4E.
• The so formed eIF-4F (referred to as cap binding protein) binds to the
cap of mRNA.
• Then elF-4A and elF-4B bind to mRNA and reduce its complex structure.
• This mRNA is then transferred to 43S complex.
• For the appropriate association of 43S preinitiation complex with
mRNA, energy has to be supplied by ATP.
• The ribosomal initiation complex scans the mRNA for the
identification of appropriate initiation codon.
• 5’-AUG is the initiation codon and its recognition is facilitated by a
specific sequence of nucleotides surrounding it. This marker sequence
for the identification of AUG is called as Kozak consensus sequences.
• In case of prokaryotes the recognition sequence of initiation codon is
referred to as Shine- Dalgarno sequence.
d) FORMATION OF 80S INITIATION COMPLEX: 48S initiation complex
binds to 60S ribosomal subunit to form 80S initiation complex.
• This binding involves the hydrolysis of GTP (bound to eIF-2). This
step is facilitated by the involvement of eIF-5.
• As the 80S complex is formed, the initiation factors bound to 48S
initiation complex are released, and recycled.
• The activation of eIF-2 requires eIF-2B (also called as guanine
nucleotide exchange factor) and GTP.
• The activated eIF-2 (i.e. bound to GTP) requires eIF 2C to form the
ternary complex.
• REGULATION OF INITIATION: The eIF-4F, a complex formed by
the assembly of three initiation factors controls initiation, and
thus the translation process.
• eif-4E, a component of eIF-4F is primarily responsible for the
recognition of mRNA cap.
• And this step is the rate limiting in translation.
• eIF-2 which is involved in the formation of 43S preinitiation
complex also controls protein biosynthesis to some extent.
• INITIATION OF TRANSLATION IN PROKARYOTES: The formation of
translation initiation complex in prokaryotes is less complicated
compared to eukaryotes.
• The 30S ribosomal subunit is bound to initiation factor 3 (IF-3) and
attached to ternary complex of IF-2, formyl met tRNA and GTP.
• Another initiation factor namely IF-I also participates in the formation of
preinitiation complex.
• The recognition of initiation codon AUG is done through Shine Dalgarno
sequence.
• A 50S ribosome unit is now bound with the 30S unit to produce 70S
initiation complex in prokaryotes.
INITIATION OF
TRANSLATION
• ELONGATION OF TRANSLATION: Ribosomes elongate the
polypeptide chain by a sequential addition of amino acids.
• The amino acid sequence is determined by the order of the
codons in the specific mRNA.
• Elongation, a cyclic process involving certain elongation factors
(EFs), may be divided into three steps: a) Binding of aminoacyl
t-RNA to A-site.
b) Peptide bond formation.
c) Translocation.
a) BINDING OF AMINOACYL tRNA to A SITE: The 80S initiation complex
contains met tRNAi in the P-site, and the A-site is free.
• Another aminoacyl-tRNA is placed in the A-site.
• This requires proper codon recognition on the mRNA and the
involvement of elongation factor 1a (EF-Ia) and supply of energy by GTP.
• As the aminoacyl-tRNA is placed in the A-site, EF-1 and GDP are
recycled to bring another aminoacyl-tRNA.
b) PEPTIDE BOND FORMATION: The enzyme peptidyl transferase
catalyses the formation of peptide bond.
• The activity of this enzyme lies on 28S RNA of 60S ribosomal subunit
• It is therefore the rRNA (and not protein) referred to as ribozyme
that catalyses the peptide bond formation.
• As the amino acid in the aminoacyl-tRNA is already activated, no
additional energy is required for peptide bond formation.
• The net result of peptide bond formation is the attachment of the
growing peptide chain to the tRNA in the A-site.
c) TRANSLOCATION: As the peptide bond formation occurs, the
ribosome moves to the next codon of the mRNA (towards 3’-end).
• This process called translocation, basically involves the movement
of growing peptide chain from A-site to P-site.
• Translocation requires EF-2 and GTP.
• GTP gets hydrolysed and supplies energy to move mRNA.
• EF-2 and GTP complex recycles for translocation.
• In case of prokaryotes, the elongation factors are different, and they
are EF-Tu, EF-Ts (in place of of EF-1a) and EF-G (instead of EF-2).
• INCORPORATION OF AMINO ACIDS IN TRANSLATION: It is estimated
that about six amino acids per second are incorporated during the
course of elongation of translation in eukaryotes.
• In case of prokaryotes, as many as 20 amino acids can be incorporated
per second.
FORMATION OF PEPTIDE BOND IN
TRANSLATION
• Thus the process of protein/polypeptide synthesis in translation occurs with
great speed and accuracy.
• TERMINATION OF TRANSLATION: Termination is a simple process when
compared to initiation and elongation.
• After several cycles of elongation, incorporating amino acids and the
formation of the specific protein/polypeptide molecule, one of the stop or
termination signals (UAA, UAG and UCA) terminates the growing polypeptide.
• The termination codons which act as stop signals do not have specific tRNAs
to bind.
• As the termination codon occupies the ribosomal A-site, the release factor
namely eRF recognises the stop signal.
• eRF-GTP complex, in association with the enzyme peptidyl transferase, cleaves
the peptide bond between the polypeptide and the tRNA occupying P-site.
• In this reaction, a water molecule, instead of an amino acid is added.
• This hydrolysis releases the protein and tRNA from the P-site.
• The 80S ribosome dissociates to form 40S and 60S subunits which are
recycled.
• The mRNA is also released.
• INHIBITORS OF PROTEIN SYNTHESIS: Translation is a complex process and it
has become a favourite target for inhibition by antibiotics.
• Antibiotics are the substances produced by bacteria or fungi which inhibit the
growth of other organisms.
ELONGATION AND
TERMINATION OF
TRANSLATION
• Majority of the antibiotics interfere with the bacterial protein
synthesis and are harmless to higher organisms.
• This is due to the fact that the process of translation
sufficiently differs between prokaryotes and eukaryotes.
• STREPTOMYCIN: Initiation of protein synthesis is inhibited by
streptomycin. It causes misreading of mRNA and interferes
with the normal pairing between codons and anticodons.
• CHLORAMPHENICOL: It acts as a competitive inhibitor of the
enzyme peptidyl transferase and thus interferes with
elongation of peptide chain.
• TETRACYCLINE: It inhibits the binding of aminoacyl tRNA to the
ribosomal complex. In fact, tetracycline can also block
eukaryotic protein synthesis. This, however, does not happen
since eukaryotic cell membrane is not permeable to this drug.
• PUROMYCIN: This has a structural resemblance to aminoacyl
tRNA. Puromycin enters the A site and gets incorporated into
the growing peptide chain and causes its release. This antibiotic
prevents protein synthesis in both prokaryotes and eukaryotes.
• ERYTHROMYCIN: It inhibits translocation by binding with 50S
subunit of bacterial ribosome.
• DIPHTHERIA TOXIN: It prevents translocation in eukaryotic protein
synthesis by inactivating elongation factor eEF2.
4. CHAPERONES AND PROTEIN FOLDING: The three dimensional
conformation of proteins is important for their biological functions.
• Some of the proteins can spontaneously generate the correct functionally
active conformation, ex: denatured pancreatic ribonuclease.
• However, a vast majority of proteins can attain correct conformation, only
through the assistance of certain proteins referred to as chaperones.
• Chaperones are heat shock proteins (originally discovered in response to
heat shock).
• They facilitate and favour the interactions on the polypeptide surfaces
to finally give the specific conformation of a protein.
• Chaperones can reversibly bind to hydrophobic regions of unfolded
proteins and folding intermediates.
• They can stabilise intermediates, prevent formation of incorrect
intermediates, and also prevent undesirable interactions with other
proteins.
• All these activities of chaperones help the protein to attain compact
and biologically active conformation.
• TYPES OF CHAPERONES: Chaperones are categorised into two major
groups.
• Hsp70 SYSTEM: This mainly consists of Hsp70 (70 kDa heat shock
protein) and Hsp40 (40 kDa Hsp).
• These proteins can bind individually to the substrate (protein) and
help in the correct formation of protein folding.
• CHAPERONIN SYSTEM: This is a large oligomeric assembly which
forms a structure into which the folded proteins are inserted.
• The chaperonin system mainly has Hsp60 and Hsp10 i.e. 60 kDa
Hsp and 10 kDa Hsp.
• Chaperonins are required at a later part of the protein folding
process, and often work in association with Hsp70 system.
• PROTEIN MISFOLDING AND DISEASES: The failure of a protein to fold
properly generally leads to its rapid degradation.
• Cystic fibrosis (CF) is a common autosomal recessive disease.
• Some cases of CF with mutations that result in altered protein (cystic
fibrosis transmembrane conductance regulator or in short CFTR) have
been reported.
• Mutated CFTR cannot fold properly, besides not being able to get
glycosylated or transported. Therefore, CFTR gets degraded.
• Certain neurological diseases which are due to cellular accumulation of
aggregates of misfolded proteins or their partially degraded products
have been identified.
• The term prions (proteinous infectious agents) is used to collectively represent
them.
• Prions exhibit the characteristics of viral or microbial pathogens and have been
implicated in many diseases, ex: mad cow disease, Creutzfeldt-Jacob disease,
Alzheimer’s disease, Huntington’s disease.
5. POST- TRANSLATIONAL MODIFICATION OF PROTEINS: The proteins
synthesised in translation are, as such, not functional.
• Many changes take place in the polypeptides after the initiation of their
synthesis or, most frequently, after the protein synthesis is completed.
• These modifications include protein folding, trimming by proteolytic
degradation, intein splicing and covalent changes which are collectively known
as post-translational modifications.
• PROTEOLYTIC DEGRADATION: Many proteins are synthesised as the
precursors which are much bigger in size than the functional proteins.
• Some portions of precursor molecules are removed by proteolysis to
liberate active proteins. This process is commonly referred to as trimming.
• The formation of insulin from preproinsulin, conversion of zymogens
(inactive digestive enzymes, ex: trypsinogen) to the active enzymes are
some examples of trimming.
• INTEIN SPLICING: Inteins are intervening sequences in certain proteins.
These are comparable to introns in mRNAs.
• Inteins have to be removed, and exteins ligated in the appropriate order
for the protein to become active.
• COVALENT MODIFICATIONS: The proteins synthesised in translation
are subjected to many covalent changes.
• By these modifications in the amino acids, the proteins may be
converted to active form or inactive form.
• PHOSPHORYLATION: The hydroxyl group containing amino acids of
proteins, namely serine, threonine and tyrosine are subjected to
phosphorylation. The phosphorylation may either increase or
decrease the activity of the proteins.
• A group of enzymes called protein kinases catalyse phosphorylation
while protein phosphatases are responsible for dephosphorylation,
ex: glycogen synthase.
• HYDROXYLATION: During the formation of collagen, the amino acids
proline and lysine are respectively converted to hydroxyproline and
hydroxylysine. This hydroxylation occurs in the endoplasmic reticulum
and requires Vitamin C.
• GLYCOSYLATION: The attachment of carbohydrate moiety is essential
for some proteins to perform their functions.
• The complex carbohydrate moiety is attached to the amino acids,
serine and threonine (O-linked) or to asparagine (N-linked), leading to
the synthesis of glycoproteins.
• Vitamin K dependent carboxylation of glutamic acid residues in
certain clotting factors is also a post-translational modification.
AN OUTLINE OF POST-TRANSLATIONAL
MODIFICATION OF PROTEINS
POST-TRANSLATIONAL MODIFICATIONS OF
PROTEINS THROUGH THEIR AMINO ACIDS