Last Update: 7 December 2017 Part I

M 49
PROTEIN SYNTHESIS (TRANSLATION)
(Ref: G.Karp, Baltimore, Gene - VI & De Robertis).

Protein synthesis or translation, is the most complex synthetic activity occurring in the cell. Whereas
other macromolecules of the cell are manufactured as a result of relatively straightforward enzymatic
reactions, the assembly of a protein requires various components like -
i) All the families of tRNAs with their attached aminoacids acts as molecular adaptor.
ii) Ribosomes - gives the site for protein synthesis.
iii) Messenger RNA, - encodes the genetic information copied from DNA in the form genetic code.
iv) A number of proteins factors having a variety of functions.
v) Cations, - act as catalyst and
vi) GTP - provides energy by break down/hydrolysis
The complexity is not surprising considering that protein synthesis requires the incorporation of
each of 20 different amino acids in the precise sequence dictated by a coded messege written in a
language that uses different symbols.
Translation refers to the whole process by which the base sequence of an m RNA is used to
order and to join the amino acids in a protein. Because the linear order of amino acids in each protein
determines its function, the mechanism that maintains this order during protein synthesis is critical. The
three types of RNA (m RNA and t-RNA) participate in this essential protein synthesizing pathway in all
cells and perform different but cooperative functions, probably the molecular key to the origin of life.
Translation mechanism:
The synthesis of polypeptide chain can be divided into three, rather distinct activities :
Initiation of the chain
involves the reactions that
preceds formation of the
peptide bond between the
first two aminoacids of the
protein. It requires the
ribosome to bind to the m
RNA, forming an
initiation complex that
contains the first
aminoacyl-t RNA. This is
a relatively slow step in
protein synthesis, and
usually determines the rate
at which an mRNA is
translated.
Elongation of the chain
includes all the reactions
from synthesis of the first
peptide bond to addition
of the last amino acid.
Amino acids are added to
the chain one at a time, the
addition of an amino acid

1
 is the most rapid step in protein synthesis.
Fig : Aminoacylation of tRNA
(The two-step reaction requires energy from the hydrolysis of ATP. The equilibrium of overall reaction favors the indicated
products because the pyrophosphate (PPi) released in Step1 is converted to inorganic phosphate (Pi) by a pyrophosphase.
A=Adenine, C=Cytosine).
Termination of the chain encompasses the steps that are needed to release the completed polypeptide
chain, at the same time, the ribosome dissociates from the m RNA.
The mechanism of translation is described as they operate in bacterial cells. The process is
remarkably similar in eucaryotic cells. The primary difference is that translation in eucaryotic cells
involves a large number of soluble (nonvibosomal) protein factors.
Activation of Amino Acid : Charging of t-RNAs
It is critically important during protein synthesis that each t RNA molecule is attached to the
"correct" (cognate) amino acid. Amino acids are covalently linked by the cooH group to the 3' ends of
their cognate t RNA(s) by an enzyme called an amino acyl - t RNA synthetases (ARSs). The fidelity of
protein synthesis depends on the correct aminoacylation of t-RNA.
Each amino acid is recognized by a specific amino-acyl-t RNA synthetase which is capable of
"charging" all the t RNAs that are appropriate for that amino acid.
Aminoacyl t RNA synthetase provide an excellent example of the specificity of protein nucleic
acid interactions.
In the coupling reaction the enzyme links an amino acid to the free 2' or 3' hydroxyl of the ribose
of the adenosine at the 3' - terminus of t RNA molecules.
This two step reaction requires the cleavage of an ATP molecule :-
2
Enzyme + amino acid + ATP Mg enzyme(amino-acyl AMP) + Ppi (1)
t RNA + enzyme (amino acyl AMP) amino acyl t RNA + AMP + enzyme (2)
In the first step, the energy of ATP is used to "activate" the amino acid by forming an adenylated
aminoacid, which is bound to the enzyme. This is the primary energy-requiring step in the entire process
of polypeptide assembley.

Table Classification of Amino acyl- t RNA Synthetases (ARSs) The or in parentheses refer to the
subunit composition of each enzyme, with and designating different polypeptides. The sequences of
the and chairs in the various enzymes are different.

Class I Synthetases (2-OH) Class II Synthelases (3-OH)

Glu () Gly (22)
Gln () Ala (4)
Arg () Pro (2)
Val () Ser (2)
Ile () Thr (2)
Leu () Asp (2)??
Met (2) Asn (2)
Tyr (2)?? His (2)
Trp (2)? Lys (2)

In the second step, the amino acyl group is transferred from the enzyme complex to the t RNA
with release of AMP.
About half the ARSs transfer the aminoacyl group to the 2' hydroxyl of the terminal adenosine
(Class-I) of t RNAs and about half, to the 3' hydroxyl (class-II). The resulting aminoacyl-t RNA retains
the energy of the ATP, and the amino acid residse is said to be activated.
The overall reaction is-
Amino acid + ATP + t RNA enzyme amino acyl t RNA + AMP + 2 Pi
The equilibrium of the reaction is driven further toward activation of the amino acid because pyro
phosphatase then splits the high-energy phosphoanhydride bond in pyrophosphate (PPi Pyrophosph
atase

2pi).

2
 From the above discussion it is apparent that the aminoacid itself plays no direct role in determing
where it is placed in the polypeptide. Rather, the codons of the m RNA are interpreted according to the
recognition abilities of the aminoacyl t RNA synthetases. The enzyme is so specific to react with an
appropriate amino acid and then to transfer this amino acid to specific t RNA by the second reaction. This
"proofreading" mechanism of the enzyme is an essential feature in the translation of the genetic code.

Fig : The N-formyl-methionyl-t

Met
RNA (f Met- i RNA f ) is generated
by formylation of Methionyl-t RNA,
using formyl- tetra hydrofolate as
cofactor.

Initiation events in Translation:

The first event of the initiation stage is attachment of a free molecule of methionine (Met) to the
end of a t RNAMet by a specific aminoacyl-t RNA synthetase. There are at least two types of t RNAMet.
One designated t RNA Met that can initiate protein synthesis, and another that can incorporate
methionine within growing protein chain. The same enzyme, methionyl t RNA synthetase (Met Rs), can
attach methionine to both t RNAs, but only methionyl t RNAMet (i.e. activated methionine attached to t
RNA Met) can bind to small ribosomal sub-unit to begin the process of protein synthesis. (In bacteria, the
amino group of the methionine of methionyl - t RNA Met is modified by the addition of a formyl group
and is sometimes designated N-formylmethionyl - i RNA Met f . However, methionyl-t RNA iMet
[abbreviated Met-t RNA iMet] is commonly used to designate the initiator t RNA in all cases).
In the second step the Met-t RNAi Met (assisted by a protein - GTP complex) and a small
ribosomal sub-unit bind to the m RNA at a specific site most often located quite near to the first (or one
of the first) AUG sequence in the messege, which serves as the initiation codon. GUG in some bacteria
may also acts as initiation codon and codes for methionine, whereas when anywhere else in coding
sequence it specifies valine. This is example of site-specific variation in codon translation.
In most bacteria, the small ribosomal sub-unit identifies initiation sites through the interaction of
short nucleotide sequences in the small 16 s r RNA and the m RNA. On the m RNA a specific sequence
of nucleotides, celled Shine-Dalgarno sequence after its discoverers, resides 5 to 10 nucleotides before
the initiation cod on (near a protein start site. The shine Dalgamo sequence is complementary to a
sequence of nucleotides near the 3' end of the 16 s ribosomal RNA molecules.
Thus
Every protein start signal does not match this r RNA sequence exactly, but on average six out of
eight nucleotides match. Thus bacterial r RNA plays a direct role in recruiting a ribosome to a protein
start site on the m RNA.
In eucaryotic cells the mechanism by which a small ribosomal subunit finds start sites is not fully
understood. Almost all eucaryotic m RNAs have a single start site at which synthesis of a single protein
begins. The first signal thought to be recognized is the 5' cap present on all eucaryotic m RNAs. Usually,
after cap recognition, the bound ribosomal subunit then is thought to slide along the m RNA to locate an
AUG. Frequently the first AUG is used, but the presence of certain nucleotides surrounding the initiating

3
AUG greatly increases the effectiveness of initiation. This sequence, referred to as a kozak sequence, for
Marilyn Kozak, who showed its importance, is
m RNA 5' - ACC AUGG-
It is generally agreed that initiation of translation of most eucaryotic m RNAs involves
recognition of the cap followed either by use of the first downstream AUG or by the locating of a 5'-
proximal AUG with a consensus sequence surrounding the AUG codon.
Initiation factors : A group of soluble proteins called initiation factors (IFs in procaryotes and eIFs in
eucaryotes) help the small ribosomal sub-unit find the initiation site.
Three initiation factors (IF1, IF2 and IF3) have been characterized from procaryotic cells, and at
least five (some of which have several protein components) are known in eucaryotic cells.
In procaryotes, IF3 is critical in finding the AUG. In eucaryotes, the large eIF4 complex helps to
ensure that the 5' end of the m RNA is single stranded (i.e. the factor serves both to bind the cap and to
unwind any secondary structure that may exist); it also ensures that the 5' end is ready for the small sub-
unit to locate the AUG. Only after the small subunit binds at the AUG is the large sub-unit added to the
complex.

Fig : Fonrmation of Initiation Complex in

Procaryotes (Ret.- Baltimore)

In bacteria, three initiation factors needed

for both m RNA and t RNA to enter the
initiation complex.
IF3 is needed for 30s sub-units to bind
specifically to initiation sites in m
RNA.
IF2 binds a special initiator t-RNA and
controls its entry into ribosome in
response to first AUG codon which
requires hydrolysis of GTP. Positioning
of larger sub-unit (50s) of ribosome
also controls by IF2. GTP complex.
GTP hydrolysis probably drives a
conformational change in the ribosome
that is required for initiating
translation.
IF1 binds to 30s sub-units only as a
part of the complete initiation complex
and could be participating in
interaction between IF2 and initiator t RNA. It may also involves in stabilizing the complex.
The IF3 has dual functions in initiation reaction. It is needed first to stabilize (free) 30s sub-units
acting as ribosome dissociation factor and then it enables them to bind in RNA. IF3 essentially controls
the freedom of 30S sub-units, which lasts from their dissociation from the pool of ribosomes to their
reassociation with a 50S sub-unit at initiation.
The first function of IF3 controls the equilibrium between ribosomal states. IF3 binds to free 30s
sub-units that are released from the pool of 70s ribosomes. The presence of IF3 prevents the sub unit
from reassociating with a 50S subunit, thereby acting as an antiassociation factor, that causes a 30S sub-
unit to remain in the pool of free subunits. IF3 must be released from the 30s m RNA complex after
formation in order to enable the 50s sub-unit to join. On its release, IF3 immediately recycles by finding
another 30S sub-unit.

4
 Fig : Initiation Complex
formation in Eucaryotes. (Ref.
Baltsmore)

Eucaryotic initiation proceeds through the formation of a ternary complex containing Met-t RNA Met,
eIF2 and GTP. The complex is formed in two stages. GTP binds to eIF2 and this increases the factors
affinity for Met-t RNAi Met which then is bound. The ternary complex then associates directly with free
40s sub units. The reaction is independent of the presence of m RNA. In fact, the Met-t RNAi Met
initiator must be present in order for the 40S sub unit to bind to m RNA.
When the small subunit has bound m RNA, it migrates to (usually) the first AUG codon. It
requires expenditure of energy in the form of ATP. When the small sub-unit reaches the initiation site,
it stops, and can be joined by a large sub-unit (60s) to form complete ribosome (80s).
The eIF4 factor is a multimer of several protein, that reognizes the cap, and eIF4A unwinds any
secondary structure that exists in the first 15 bases of the m RNA. Energy for unwinding is provided by
ATP. Unwinding of structure farther along the m RNA is accomplished by eIF4A together with another
factor, eIF4B.

5
 Factors eIF3 and eIF6 are required to maintain sub-units in their dissociated state. The eIF6binds
to the large sub-unit which it is released then large sub-unit joins the initiation complex.
Junction of the 60s sub-units with the initiation complex can not occur until eIF2 and eIF3 have
been released from the initiation complex, a function mediated by eIF5, which is a GTP ase. Probably all
of the remaining factors are released
when the complete 80s ribosome is
formed.
Phosphorylation of eIF2
controls the rate of protein synthesis
in eucaryotes. Phosphorylated eIF2 is
inactive in protein synthesis, and the
degree of phosphosylation can be
regulated by cyclic AMP in a cascade
mechanism.
Ribosome sites for t RNA binding :
Ribosomes provide three t
RNA binding sites (A,P and E) during
protein synthesis. The A site is the
incoming site or acceptor site into
which aminoacyl-t RNAs enter the
ribosome- m RNA complex.
The P site (for peptidyl-t
RNA) is called the donar site where
peptide bonds are formed and t RNA
donates aminoacids to the growing
chain.
A third site, the E site is
transiently occupied by the deacylated
end of the t RNA that has completed
amino acid transfer and is about to be
rejected.
As the contributor of the first
amino acid of the chain, the Met-t
RNAi Met enters the P position.

Fig : Elongation and termination of Polypeptide Synthesis (Ref- Battimore)

Elongation events in Translation :

Once protein synthesis has been initiated, additional factors are required for the elongation of the
peptide chain, called elongation factors.
The second amino acid is bound to the A site of the ribosome by a specific aminoacyl t RNA with
the help of elongation factor EFTu in procaryotes and eEF1 in eucaryotes linked to GTP. EF-Tu is
used to carry all other aminoacyl - t RNAs to the site of chain elongation. Once the proper amino acyl - t
RNA-Tu-GTP (ternary complex) is bound to the m RNA codon, the GTP is hydrolzed and the Tu-GDP
(binary complex) complex released to work again with another amino acyl - t RNA. Thus it displays the
cyclic association with, and dissociation from, the ribosome that is the hallmark of the accessory factors.
When aminoacyl-t RNA has been placed in the A site, codon-anticodon recognition triggers a
change in the conformation of EF-Tu. Then the GTP is cleaved, and the binary complex EF-Tu. GTP is
released.
Another factor, EF-TS mediates the regeneration of the used form, EF.Tu.GDP, into the active
form, EF-TU.GTP. First, EF-TS displaced the GDP from EF-TU, forming the combined factor EF-
TU.EF-TS. Then the EF-TS is in turn displaced by GTP, reforming EF-TU.GTP. The active binary

6
complex binds amino acyl t RNA and the released EF-TS can recycle. In eucaryotes a homologous factor,
e EF-1 is responsible for activation process of e EF-1 with GTP.

The Second step in Fig : Peptide bond

formation between
the elongation cycle is the two aminoacids
formation of a peptide bond attached with two
between the amino acids tRNAs at two sites
attached to the two t RNAs of ribosome
at A and P sites of ribosome.
The reaction is accomplished
by the transfer of the
methionine (or N-
formylmethionine) on the
initiator tRNA of the P site
to the amino acid attached to
the t RNA bound to the
second codon in the A site.
The reaction is catalyzed by
peptidyl transferase, a
component of the large
subunit of the ribosome. In other wards, peptidyl transferase is a ribozyme as it resides in the large
ribosomal RNA molecules (235 r RNA) The energy for peptide bond formation is stored in the AA t
RNA and comes from ATP. The formation of the first peptide bond leaves one end of the t RNA
molecule of the A site still attached to its complementary codon on the m RNA and the other end of the
molecule attached to a dipeptide. The t RNA of the P site is now devoid of any linked amino acid.
The third step in the elongationcycle involves - (I) ejection of the uncharged t RNA from the P
site and
ii) the movement of ribosome three nucleotides (one codon) along the m RNA in the 3' direction : This
step is called translocation, is accompanied by the movement of the t RNA - dipeptide to the P site of the
ribosome, sitill hydrogenbonded to the second codon of the m RNA.
Translocation requires another elongation factor EF-G in procaryotes, or G factor and eEF2 in
eucaryotes; also called translocase. G factor binds GTP (hence its name) and carries it to the ribosome,
where it is hydrolysed to GDP. The energy realised is used for the translocation process and for the
realase of the deacylated tRNA used to translate the previous codon.
The free t RNA from the P site of the ribosome first dislocated to the E site from which it is
ejected. The A site is now unfilled and ready for the next aminoacyl t RNA.
Once the third charged t RNA is associated with the m RNA in the A site, the dipeptide from the t
RNA of the P site is transferred to the amino acid on the t RNA of the A site, forming the second peptide
bond and a tripeptide attached to the t RNA of the A site. The t RNA in the P site is once again devoid of
an amino acid. Peptide bond formation is followed by ejection of the t RNA from the P site and
translocation of the ribosome to the fourth codon, and the cycle is ready to begin again.
Termination of Translation :
The elongation process of translation continues until or unless a termination codon or stop codon
reaches the A site of the ribosome. Chain termination leads to the release of the ribosome. Chain
termination leads to the release of the free polypeptide and t RNA and to the dissociation of the ribosome
into subuints.
Out of 64 possible trinucleotide codons only 61 triplets are assigned to amino acids, the other 3
are termination eodons that ends protein synthesis. These are UAG (the amber codon), UAA (the ochre
codon) and UGA (the opal codon). When the ribosome reaches one of these codons, the signal is read to
stop further elongation and to release of the peptidyl-t RNA complex. [UGA located internally in the
coding sequence in the appropriate nucleotide sequence encoding modified aminoacid selenocystein]

7
 Termination of chain elongation requires the presence of release factors (RFs) or termination
tactors (TFs). There are three (RFI, RFII, RFIII) such factors in bacteria and only one in eucaryotes
(eRF).
In bacteria two release factors RF1 and RF2 are specific for different sequences.
RF1 recognizes UAA and UAG; and
RF2 recognizes UGA and UAA.
These release factors act at the ribosomal A site and require polypeptidyl-t RNA in the P site. The
factors are present at much lower levels than initiation or elongatioin factors, there are about 600
molecules of each per cell, equivalent to 1 RF per 10 ribosomes. The codon specific release factors are
assisted by another factor, RF-3, which is a GTP binding protein retated to EF-G.
The release factor carries a bound GTP that is subsequently hydrolyzed. Once translation stops,
the completed polypeptide is severed from its attachment to the t RNA, and both the release factor and
deacylated t RNA are released from the ribosome. Hydrolysis of the bond between the polypeptide and
the last t RNA may be catalyzed by the same part of the r RNA responsible for peptide bond formation
during elongation. Once termination is completed, the ribosome separates from the messege and
dissociates into its subunits in preparation for another round of translation.

Fig : Schematic drawing of Polyribosome.

Mechanisms of eucaryotic release factors (eRF) are less well characterized. Since the three
termination codons can be readily formed by single base changes from many other codons, one might
expect mutations to arise that produce stop codons within a gene. Such mutations would result in the
premature termination of the growing polypeptide chain. Mutations of this type termed nonsense
mutations, but it is really a misnomer since the codons do have meaning albeit a disruptive one in a
mutant gene. Variety of inherited diseases are caused by these mutations in human because only a portion
of the protein is synthesized and thus is invariably non functional.
Formation of Polyribosome :
When a messenger RNA in the process of being translated is examined in the electron
microscope, a number of ribosomes are invariably seen to be attached along the length of the m RNA
thread. This complex of ribosomes and m RNA is called a polyribosome, or polysome. Each of the
ribosome initially assembles from its subunits at the initiation codon and then moves from that point
toward the 3' end of the m RNA until it reaches a termination codon. As soon as each ribosome moves a
sufficient distance along the messege from the initiation codon, the next ribosome attaches to the m RNA
and begins its translation activity. The simultaneous translation of the same m RNA by numerous
ribosomes greatly inereases the rate of protein synthesis within the cell.
Protein Translocation :
Eucaryotic cells have evolved complicated cellular organelleles in order to ensure that proteins
reach their appropriate destination. Some proteins are translocated during synthesis into the lumen of
rough endoplasmic reticulum, from which they are channeled via coated vesicles into the Golgi
apparatus, where they are glycosylated and targeted to secretary grannles, membranes or lysosomes.
Some protein remain free in the cytosol in quasi-soluble form, others associated with macromolecular
cytosolic structures, such as filaments, microtubules, centrioles etc. This class also includes nuclear
proteins which pass into the nucleus through large aqueous pores.

8
 The process of inserting into or passing through a membrane is called protein translocation.
Protein that associated with membranes follow one of two routes. Mitochondrial and chloroplast proteins
are released into the cytosal and subsequently associate with the organelle membranes. Because this
process takes place after synthesis of protein has been completed, it is called post-translational
translocation.
The general process of finding its ultimate destination by transport through sucessive membrane
system is called protein sorting or protein trafficking.
The N-terminal sequence of the protein is cleaved from the protein during protein translocation as
it comprises a leader that is not part of the mature protein. The protein carrying the leader is called
preprotion.
Thus a considerable part of the cell is concerned with the synthesis and traffic of proteins. But the
effort is worth it, for once all these proteins are assembled into their proper places, they provide the
delecate biochemical machinery that keeps cells feeding, locomotion, multiplying, and alive.