TRANSLATION

MOLECULAR BIOLOGY

AVIJIT CHAKRABORTY
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The genetic code

During translation, a cell “reads” the information in a messenger RNA (mRNA) and uses it to
build a protein. Actually, to be a little more techical, an mRNA doesn’t always encode—
provide instructions for—a whole protein. Instead,
what we can confidently say is that it
always encodes a polypeptide, or
chain of amino acids.

In an mRNA, the instructions for

building a polypeptide are RNA
nucleotides (As, Us, Cs, and Gs)
read in groups of three. These
groups of three are called codons.
There are 61 codons for amino acids,
and each of them is "read" to specify
a certain amino acid out of
the 202020 commonly found in
proteins. One codon, AUG, specifies the amino acid methionine and also serves as a start
codon , signalling the start of protein synthesis. There are three more codons that do not specify
amino acids. These stop codons, UAA, UAG, and UGA, tell the cell when a polypeptide is
complete. All together, this collection of codon-amino acid relationships is called the genetic
code, because it lets cells “decode” an mRNA into a chain of amino acids.

The genetic code refers to the set of rules by which information in DNA (and RNA) is
translated into proteins. Its main characteristics are:

1. Triplet code: Each amino acid is coded by a sequence of three nucleotides, called a
codon.
2. Universal: The genetic code is almost the same in all organisms, from bacteria to
humans (with a few minor exceptions, like mitochondria).
3. Degenerate (Redundant): Most amino acids are coded by more than one codon..
Example: Leucine has several codons.
4. Unambiguous (Specific): Each codon codes for only one amino acid (or a stop
signal), never more than one.
5. Non-overlapping: Codons are read one after another, without sharing nucleotides.
6. Comma-less (Continuous): The code is read continuously, without gaps or
punctuation between codons.
7. Start and Stop codons: Start codon: AUG (codes for Methionine) Stop codons:
UAA, UAG, UGA
8. Colinear: The sequence of codons in mRNA corresponds directly to the sequence of
amino acids in the protein.
9. Polarity: The genetic code is read in the 5′ → 3′ direction on mRNA.

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Introduction to Translation in Prokaryotes:

The process by which proteins are produced with amino acid sequences specified by the
sequence of codons in messenger RNA is called translation. Translation is the first stage of
protein biosynthesis.

DNA is the master molecule which posseses the genetic information about the sequence of
amino acids in a polypeptide chain. Structure and properties of DNA regulate and control the
synthesis of proteins.

DNA present in the nucleus sends out information in the form of messenger RNA into the
cytoplasm, which is the site of the protein synthesis in eukaryotes. The messenger RNA carries
the information regarding the sequence of amino acids of the polypeptide chain to be
synthesized. This message or information is in the form of a genetic code. This genetic code
specifies the language of amino acids to be assembled in a polypeptide.

The main points about translation in prokaryotes are given below:

1. Site: Translation occurs in the cytoplasm where the ribosomes are located. Ribosomes are
made of a small and large subunit which surrounds the mRNA. In prokaryotic translation 70S
ribosomes with 30S and 50S subunits are used. The mRNA is synthesized from DNA only. In
prokaryotes, there are several initiation and termination sites.
2. Template: In translation, messenger RNA (mRNA) is decoded to produce a specific
polypeptide according to the rules specified by the genetic code. This uses an mRNA sequence
as a template to guide the synthesis of a chain of amino acids that form a protein. Many types
of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA are not
necessarily translated into an amino acid sequence.
3. Requirements: The translation process requires mRNA, rRNA, ribosomes, 20 kinds of
amino acids and their specific tRNAs.
4. Factors Involved: In prokaryotes, three factors are involved in the initiation of translation
[IF 1, IF 2 and IF 3], one factor in the elongation of polypeptide chain and three factors in chain
termination [RF1, RF2 and RF3],
5. Enzymes Involved: Two types of enzymes are used in translation. Aminoacyl tRNA
synthetase (an enzyme) catalyzes the bonding between specific tRNAs and the amino acids.
The enzyme peptidyl transferase connects A site and P site by forming a peptide bond [the
nitrogen carbon bond] during elongation phase.
6. Codons Involved: In the process of translation two types of codons, viz., start codon and
stop codons are involved. The codon, AUG, initiates the process of translation and one of three
stop codons i.e. UAA, UAG or UGA is used for chain termination.
7. Starting Amino Acid: In prokaryotes, starting amino acid is N-formyl methionine.
Moreover, there is overlapping of transcription and translation.

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Role of Ribosomes in Protein synthesis:

Ribosome is a macromolecular structure that directs the synthesis of proteins. A ribosome is a

multicomponent, compact, ribonucleoprotein particle which contains rRNA, many proteins and
enzymes needed for protein synthesis. Ribosome brings together a single mRNA molecule and
tRNAs charged with amino acids in a proper orientation so that the base sequence of mRNA
molecule is translated into amino acid 1 sequence of polypeptides. Ribosome is a nucleoprotein
particle having two subunits. These two subunits lie separately but come together for the
synthesis of polypeptide chain. In E. coli ribosome is a 70S particle having two subunits of 30S
and 50S. Their association and dissociation depends a upon the concentration of magnesium.
Small subunit of ribosome contains the decoding centre in which charged tRNAs decode o the
codons of mRNA. Large subunit contains peptidyl transferase centre, which forms the peptide
bonds between successive amino acids of the newly synthesized peptide chain.

Both 30S and 50S subunits consist of ribosomal RNA (rRNA) and proteins. The mRNA binds
to the 16S rRNA of smaller subunit. Near its 5′-end mRNA binds to the 3′-end of 16S rRNA.

The main role of ribosome is the formation of peptide bond between successive amino acids of
the newly synthesized polypeptide chain. The ribosome has two channels in it. The linear
mRNA enters and escapes through one channel, which has the decoding centre. This channel
is accessible to the charged tRNAs. The newly synthesized polypeptide chain escapes through
the other channel.

Factors Translation steps Functions

IF-1 Initiation Helps to stabilize 30S ribosomal subunit
Binds fmet-tRNA with 30S subunit mRNA
IF-2 Initiation complex; bind GTP and hydrolyse
IF-3 Initiation Binds 30S subunit with mRNA
Binds GTP; bring Aminoacyl-tRNA to A-site
EF-TU Elongation of ribosome
EF-TS Elongation Generates EF-TU
EF-G Elongation Helps in translocation of ribosome
Helps to dissociates polypeptide from tRNA
RF-1 Termination ribosome complex; specific for UAA and UAG
Helps to dissociates polypeptide; specific for
RF-2 Termination UGA and UAA
RF-3 Termination Stimulates RF-1 and RF-2
Direction of Translation:

Each protein molecule has an -NH2 end and -COOH end. Synthesis begins at amino end and
ends at carboxyl end. The mRNA is translated in 5 → 3′ direction from amino to carboxyl end.
Synthesis of mRNA from DNA transcription also occurs in 5′ → 3′ direction.

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Activation of amino acids

1. The activation of aminoacids take place in cytosol.

2. The activation of aminoacids is catalyzed by their aminoacyl tRNA synthetases.
3. All the 20 aminoacids are activated and bound to 3’ end of their specific tRNA in the
presence of ATP and Mg++.
4. The N-formylated methionine is chain initiating aminoacid in bacteria whereas
methionine is chain initiating aminoacid in eukaryotes.
5. Methionine is activated by methionyl-tRNA synthetase. For N-formylmethionine two
types of tRNA are used ie. tRNAmet and tRNAfmet.
6. Similarly, all 2o aminoacids are activated (amino acyl-AMP enzyme complex) and then
bound to their specific tRNA forming Aminoacyl tRNA.

INITIATION
This is the first phase of translation. Start or initiation codon [AUG] is responsible for initiation
of translation process.

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Initiation of translation in prokaryotes involves the assembly of the components of the

translation system which are: the two ribosomal subunits (small and large), the mRNA to be
translated, the first (formyl) aminoacyl tRNA (the tRNA charged with the first amino acid),
GTP (as a source of energy), and three initiation factors (IF 1, IF 2 and IF 3) which help the
assembly of the initiation complex.

1. The ribosome consists of three sites, the A site, the P site, and the E site. The A site is
the point of entry for the aminoacyl tRNA (except for the first aminoacyl tRNA, fMet-
tRNAfMet, which enters at the P site). The P site is where the peptidyl tRNA is formed
in the ribosome. And the E site which is the exit site of the now uncharged tRNA after
it gives its amino acid to the growing peptide chain.

2. Translation begins with the binding of the small ribosomal subunit to a specific
sequence on the mRNA chain. Initiation of translation begins with the 50S and 30S
ribosomal subunits. IF1 (initiation factor 1) blocks the A site to ensure that the IMet-
tRNA can bind only to the P site and that no other aminoacyl-tRNA can bind in the A
site during initiation, while IF3 blocks the E site and prevents the two subunits from
associating.

3. IF2 is a small GTPase which binds fmet-tRNAfMet and helps its binding with the small
ribosomal subunit. The 3′ end of the 16S rRNA of the small 30S ribosomal subunit
recognizes the ribosomal binding site on the mRNA (Shine-Dalgarno sequence or SD),
through its anti-SD sequence, 5-10 base pairs upstream of the start codon. The Shine-
Dalgarno sequence is found only in prokaryotes.
4. This helps to correctly position the ribosome onto the mRNA so that the P site is directly
on the AUG initiation codon. IF3 helps to position fMet-tRNAfmet into the P site, such
that fMet-tRNAf metinteracts via base pairing with the mRNA initiation codon (AUG).
Initiation ends as the large ribosomal subunit joins the complex causing the dissociation
of initiation factors.
5. The small subunit binds via complementary base pairing between one of its internal
subunits and the ribosome binding site. This site a sequence of about ten nucleotides on
the mRNA. It is located anywhere from 5 and 11 nucleotides from the initiating codon
[AUG],

6. After binding of the small subunit, a special tRNA molecule, called N-formyl
methionine, or fMet, recognizes and binds to the initiator codon. Then the large subunit
binds resulting in the formation of the initiation complex. As soon as the initiation
complex is formed, the fMet-tRNA occupies the P site of the ribosome and the A site
is left empty.

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7. This entire initiation process is facilitated by extra proteins, called initiation factors that
help with the binding of ribosomal subunits and tRNA to the mRNA chain.

ELONGATION (Diagram follow class note)

This is the second phase or middle phase of translation. Elongation begins after the formation
of the initiation complex. Elongation of the polypeptide chain involves addition of amino acids
to the carboxyl end of the growing chain. The growing protein exits the ribosome through the
polypeptide exit tunnel in the large subunit.

Elongation starts when the fmet-tRNA enters the P site, causing a conformational change which
opens the A site for the new aminoacyl-tRNA to bind. This binding is facilitated by elongation
factor-T4 (EF-T4), a small GTPase. Now the P site contains the beginning of the peptide chain
of the protein to be encoded and the A site has the next amino acid to be added to the peptide
chain.

The growing polypeptide connected to the tRNA in the P site is detached from the tRNA in the
P site and a peptide bond is formed between the last amino acids of the polypeptide and the
amino acid still attached to the tRNA in the A site.

This process, known as peptide bond

formation, is catalyzed by a ribozyme,
peptidyltransferase, an activity intrinsic to
the 23S ribosomal RNA in the 50S
ribosomal subunit. Now, the A site has
newly formed peptide, while the P site has
an unloaded tRNA (tRNA with no amino
acids). In the final stage of elongation,
translocation, the ribosome moves 3
nucleotides towards the 3′ end of mRNA.

Since tRNAs are linked to mRNA by codon-

anticodon base-pairing, tRNAs move
relative to the ribosome taking the nascent
polypeptide from the A site to the P site and
moving the uncharged tRNA to the E exit
site.

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This process is catalyzed by elongation factor G (EF-G). The ribosome continues to translate
the remaining codons on the mRNA as more aminoacyl-tRNA binds to the A site, until the
ribosome reaches a stop codon on mRNA(UAA, UGA, or UAG).

When the A site opens again, the next appropriate aminoacyl tRNA can bind there and the same
reaction takes place, yielding a three-amino acid peptide chain. This process repeats, creating
a polypeptide chain in the P site of the ribosome. A single ribosome can translate 60 nucleotides
per second. This speed can be vastly augmented when ribosomes unite together to form
polyribosomes.

TERMINATION

1. The peptide bond

formation and elongation of
polypeptide continues until
stop codon appear on A-site.
2. If stop codon appear on A-
site it is not recognized by t-
RNA carrying aminoacids
because stop codon donot have
anticodon on mRNA.
3. The stop codon are
recognized by next protein
called release factor (Rf-1, RF-
2 and RF-3) which hydrolyses
and cause release of all
component ie 30s, 50S, mRNA and polypeptide separates.
4. RF-1 recognisaes UAA and UAg while RF-2 recognises UAA and UGA while RF-3
dissociate 30S and 50S subunits.
5. In case of eukaryotes only one release actor eRF causes dissociation.

EUKARYOTIC TRANSLATION
The broad outlines of eukaryotic protein synthesis are the same as in prokaryotic protein
synthesis. The genetic code is generally the same (some microorganisms and eukaryotic
mitochondria use slightly different codons), rRNA and protein sequences are recognizably
similar, and the same set of amino acids is used in all organisms. However, specific differences
exist between the two types of protein synthesis at all steps of the process.
Initiation
Both prokaryotes and eukaryotes initiate protein synthesis with a specialized methionyl‐tRNA
in response to an AUG initiation codon. Eukaryotes, however, use an initiator met—

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tRNA met I—that is not formylated. Recognition of the initiator AUG is also different. Only one
coding sequence exists per eukaryotic mRNA, and eukaryotic mRNAs are capped. Initiation,
therefore, uses a specialized cap‐ binding initiation factor to position the mRNA on the small
ribosomal subunit. Usually, the first AUG after the cap (that is, 3′ to it) is used for initiation.
Elongation
Most differences in elongation result from the fact that the eukaryotic cell has different
compartments, which are separated by membranes. Both prokaryotic and eukaryotic cells, of
course, have an inside and outside; however, eukaryotic proteins can be targeted to, for
example, the mitochondrion.
Translating ribosomes in eukaryotes are located in different places in the cell depending on the
fate of their proteins. Free polysomes are in the cytoplasm and synthesize cytoplasmic proteins
and those that are bound for most intracellular organelles, for example, the nucleus. Members
of the second class of polysomes, membrane‐bound polysomes, are attached to the endoplasmic
reticulum (forming the rough ER), and synthesize exported proteins. In cells that are actively
secreting enzymes or hormones (for example, those in the pancreas), most of the protein
synthesis occurs on the rough ER.
The messages encoding exported proteins must be recognized. For example, the digestive
proteases are made in the pancreas. If they are released into the pancreas rather than the
intestine, they will self‐digest the organ that makes them. Carrying out this export efficiently
is obviously important. The signal hypothesis explains how proteins destined for export are
discriminated. Proteins that are destined for export contain a short (less than 30 amino acids
long) sequence made up of hydrophobic amino acids at their amino terminus. Because peptide
synthesis occurs in the amino‐to‐carboxy direction, the signal peptide is the first part of the
protein that is made. Signal peptides are not found in most mature secreted proteins because
they are cleaved from the immature proteins during the secretion and maturation process.
The process of protein export involves a small, cytoplasmic ribonucleoprotein particle
(the Signal Recognition Particle or SRP) with the signal coding mRNA sequence and/or the
signal peptide itself. This interaction stops translation of the protein. Then, the stalled or
arrested ribosome moves to the endoplasmic reticulum (ER). A receptor on the ER binds the
SRP.
Once the complex containing ribosome, mRNA, signal peptide, and SRP is “docked” onto the
membrane, SRP leaves the complex and the ribosome resumes translation. The signal peptide
inserts across the membrane; this insertion is dependent on the hydrophobic nature of the signal
sequence. The rest of the protein follows the signal sequence across the membrane, like thread
through the eye of a needle. The protein folds into its secondary and tertiary structure in
the lumen (inside cavity) of the ER. The signal sequence is cut away from the protein either
during translation (cotranslational processing) or, less often, after the protein is released from
the ribosome (posttranslational processing). After the polypeptide chain is completed, the
ribosome is released from the ER and is ready to initiate synthesis of a new protein. Secreted
proteins are also made by prokaryotes by using a signal sequence mechanism, with the cell
membrane taking the place of the ER membrane.
This scheme can also accommodate the synthesis of membrane‐bound proteins. In this case,
the protein is not released into the lumen of the ER, but rather stays bound to the membrane.

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One or more anchor sequences (or “stop‐transfer sequences”) in the newly made protein keeps
the partially folded region of the protein in the membrane.
Protein glycosylation

Many eukaryotic membrane‐bound and secreted proteins contain a complex of sugar residues
bound to the side chains of either asparagine to make N‐linked sugar residues or serine and
threonine to make O‐linked sugar residues. The core of the glycosyl complex is assembled,
not by adding sugars one after another to the protein chain, but by synthesizing the core
oligosaccharide on a membrane lipid, dolichol phosphate.

Dolichol is a long chain of up to twenty 5‐carbon isoprenoid units. The core oligosaccharide
assembled on dolicol phosphate ultimately contains 14 saccharides that are transferred all at
once to an asparagine residue on the protein. After the glycoprotein is assembled, the core
oligosaccharide is trimmed by removal of the three glucose residues from the end, making
a high‐mannose oligosaccharide on the protein. The high‐mannose oligosaccharide is moved
from the ER for further modification in the Golgi complex, a structure composed of layered
intracellular membranes. The completed glycoprotein moves to the plasma membrane in a
membrane granule, which buds off from the Golgi. The granule fuses with the plasma
membrane to release its contents. Binding to receptors in the plasma membrane brings some
lysosomal enzymes back into the cell. The enzyme‐receptor complex is taken into the cell in a
process that is essentially the reverse of secretion, although it involves different membrane
proteins.
Fidelity in protein synthesis
The flow of genetic information from DNA to RNA to protein constitutes the basis for cellular
life. DNA replication, transcription and translation, the processes through which information
transfer occurs, are the result of millions of years of evolution during which they have achieved
levels of accuracy and speed that make modern life possible. All three processes have base
complementarity at the core of their mechanisms. DNA replication and transcription both
depend on complementarity of the incoming nucleotide to the DNA template, whereas
translation depends on the complementarity of the anticodon of the incoming transfer RNA
(tRNA) to the codon in the template messenger RNA (mRNA). Fidelity of genetic information
transfer thus relies heavily on discrimination between complementary, Watson-Crick (and in a
few cases wobble) base pairs and non-complementary ones.
To ensure high selectivity, the macromolecular machines that carry out replication,
transcription and translation — DNA polymerase, RNA polymerase and the ribosome,
respectively — have evolved specific substrate recognition strategies. These strategies exploit
the stability arising not only from the hydrogen-bonding and stacking capacity of Watson-Crick
base pairs but, more importantly, from their distinct geometry. Both polymerases and the
ribosome have chemical groups that directly monitor the geometry of the template-substrate
base pair. In the case of DNA polymerases, this ‘geometric selection’ is estimated to contribute
three orders of magnitude or more to selectivity, while hydrogen bonding only provides 7–40-
fold selectivity.

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While the accuracy of DNA replication and transcription depend only on cognate base pair
selection, translation depends on an additional, base-pairing-independent reaction that must be
carried out with high specificity. Each tRNA must be covalently attached to a specific amino
acid — aminoacylated — preserving an unambiguous codon-amino acid correspondence
known as the genetic code. This reaction is carried out by aminoacyl-tRNA synthetases specific
for each amino acid and a corresponding group of tRNAs (isoacceptors). These enzymes must
therefore recognize two substrates: first, a group of tRNAs which share a collection of ‘identity
elements’ and second, an amino acid that may be distinguished by small differences in side-
chain properties.
Polymerases, synthetases and the ribosome have been the paradigm cases for studying enzyme
specificity, though we still do not fully understand the strategies used for high fidelity
polymerization. In general terms, the specificity of an enzyme is limited by the difference in
free energy of binding between correct and incorrect substrates. This difference derives from
molecular distinctions that allow the correct substrate to make more favorable interactions with
the enzyme or enzyme-template complex. This limitation to specificity becomes a problem
during genetic information flow, where differences in free energy between cognate and some
non-cognate substrates are generally smaller than would be necessary to account, in a single
step, for the observed high fidelity of the process.
To this limitation is often added a requirement for high speed, which generally precludes full
exploitation of available free energy differences. These constraints have guided the evolution
of enzymes involved in information transfer to reach optimum ratios of accuracy and speed.
Below we discuss, primarily in the context of translation, three strategies that macromolecular
machines have evolved to achieve this balance, focusing on mechanisms known as editing,
kinetic proofreading and induced fit.
Protein synthesis or translation has an observed fidelity of 1 error in 10 3–104 polymerized
amino acids. These infrequent errors are generally substitution or missense errors resulting
from mistakes in one of two different steps during the translation process (Figure 1): first,
misacylation of a tRNA by its aminoacyl-tRNA synthetase; and second, ‘selection’ of an
incorrect tRNA during the elongation cycle. Different constraints for speed and in available
discriminatory binding energy have shaped the evolution of these two steps in translation to
achieve the necessary level of fidelity. It should be noted that other types of error, such as
incorrect start site selection, frameshifting and inappropriate termination can also occur during
translation but are not discussed here as they have been less well studied.

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Aminoacyl-tRNA synthetases use editing

Correct aminoacylation depends on the selection of two appropriate substrates, the tRNA and
the amino acid, by the corresponding aminoacyl-tRNA synthetase. tRNA selection itself
appears not to present a major challenge, as tRNAs are big enough to contain a large number
of ‘identity elements’, or determinants, for specific interactions. Amino acids, however, are
smaller and must be distinguished solely by the nature of their side-chains. Although there are
substantial chemical differences among most amino acids, the very similar chemical and/or
structural properties of some make them difficult to distinguish. As a specific example,
threonyl-tRNA synthetase must discriminate threonine from the isosteric valine and from
serine, which is smaller but has a γ-hydroxyl group like threonine.
How do synthetases deal with this? The aminoacylation reaction, which takes place at a site of
the enzyme called the synthetic site, occurs in two steps. First the amino acid is activated by
adenylation (consuming ATP) and then it is transferred to the tRNA (releasing AMP). Steric
exclusion of amino acids with larger side-chains and recognition of specific properties of each
amino acid generally make this synthetic site specific enough so that only the correct amino
acid can be activated and transferred. But amino acids having similar properties to and a smaller
size than the cognate amino acid can be misactivated at frequencies that are too high to maintain
an unambiguous code. As a consequence, enzymes facing this problem have evolved a second
active site, distinct from the synthetic site, called the editing site, where misactivated amino
acids or misacylated tRNAs are hydrolyzed.
The presence of
two catalytic sites
with different
activities led to the
proposal of a
‘double-sieve’
model of fidelity
(Figure 2). In this
model, the
synthetic site of
the enzyme acts as
the first sieve,
excluding amino acids that are too large or that cannot establish specific interactions. For
example, threonyl-tRNA synthetase can discard amino acids larger than threonine, based on
size. It also discards valine, which is similar in size but lacks the γ-hydroxyl group. Threonyl-
tRNA synthetase binds valine significantly more weakly than threonine because a specific
interaction between a zinc ion in the active site and the γ-hydroxyl group of threonine does not
form when valine is bound. Smaller amino acids that can establish sufficient interactions,
however, may slip through this first, coarse sieve and be activated and transferred to the tRNA.

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Kinetic proofreading during tRNA selection is made possible by the fact that aminoacyl-tRNAs
are delivered to the ribosome in a ternary complex with the GTPase elongation factor Tu (EF-
Tu in bacteria, EF1A in eukaryotes) and GTP. In an encounter between ternary complex and
the ribosome (initial selection), a cognate ternary complex is more likely to trigger GTP
hydrolysis than to dissociate, whereas a near-cognate ternary complex is more likely to
dissociate. Simply put, the cognate species partitions forward in the stepwise scheme whereas
the near-cognate partitions backward. This initial selection step is followed by the proofreading
step where inherent binding differences between codon and anticodon are again sampled. As
before, the cognate aminoacyl-tRNA species is more likely to partition forward (and
‘accommodate’ into the A site and participate in peptide bond formation), while the near-
cognate aminoacyl-tRNAs are more likely to partition backward (and be rejected from the
ribosome).
The relative contribution of each of these selective steps, initial selection and proofreading, has
been measured in vitro in multiple ways, where overall error rates of ~1 in 450 to 1 in 1600
approach the overall fidelity measured in vivo. In these systems, essentially all non-cognate
aminoacyl-tRNAs are rejected during initial selection. Near-cognate aminoacyl-tRNAs,
however, can pass through initial selection and trigger GTP hydrolysis with a frequency of ~1
in 30. These sneaky aminoacyl-tRNAs are generally rejected during the second stage, thus
increasing selectivity by ~15–45-fold.
Interestingly, the maximal theoretical selectivity of kinetic proofreading is not realized here
because of the processive nature of translation and associated requirement for speed. To
maximize each selective step, the forward rates should be slow enough that differences in
dissociation rates can be exploited. Indeed, experimental evidence shows that, when GTP
hydrolysis is made extremely slow, the selectivity observed in this initial selection step is
substantially increased. It was long ago suggested that ribosome mutations which affect the
fidelity of tRNA selection act similarly by increasing or decreasing the rates of individual steps
in the selection process.
While separating the process into two steps — kinetic proofreading — does provide an
advantage during tRNA selection, it is not because differences in dissociation rates between
cognate and near-cognate aminoacyl-tRNAs are exploited twice, as previously thought. Rather,
during each stage of tRNA selection a second strategy comes into play that introduces a large
difference in the rates of two critical forward steps. During initial selection, the rate of GTPase
activation (k3) is significantly faster for the cognate than for near-cognate aminoacyl-tRNAs,
and during proofreading, there are similar differential rates of accommodation (k 5) (Figure 3).

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These differences in forward rates have been attributed to a mechanism historically termed
induced fit, which is used by the translation machinery, polymerases and a number of other
enzymes. Induced fit refers to the ability of a correct substrate, but not an incorrect one, to
cause conformational changes in the enzyme and/or the substrate which have downstream
effects on catalysis. During tRNA selection on the ribosome a series of conformational changes
induced by binding of the cognate aminoacyl-tRNA, but not a near-cognate one, result in a
number of rearrangements in the ribosome and the tRNA itself that result in the kinetic effects
discussed above.
It appears then that the combination of kinetic proofreading and induced fit in tRNA selection
provides a suitable balance between fidelity and rapid elongation rates. If we take the simplest
case of kinetic proofreading, where there are no differences in forward rate constants
introduced by induced fit — where k3 and k5 are equivalent for both cognate and near-cognate
tRNAs — significant discrimination between cognate and near-cognate tRNAs will only be
observed when k3 and k5are very, very slow relative to k–2 and k7. In other words, if tRNA
selection were an equilibrium process where the full discrimination potential was extracted
from the binding energy, there would be no need for other discriminatory mechanisms. But
such a slow step in translation is apparently not compatible with the overall rapid rate of
elongation.
The addition of induced fit to the process of tRNA selection boosts selectivity when the
reaction is constrained to be fast by accelerating the rate of passage of cognate species relative
to near-cognate ones. Because forward rates are fast relative to reverse ones, the selectivity of
each step is lower than the theoretical maximum allowed by intrinsic energetic differences
between cognate and near-cognate tRNAs in the complex. In this case, the energetic cost of
inducing conformational changes has little impact on cognate tRNA selection but has
substantial detrimental effects on near-cognate tRNA selection thus conferring increased
specificity. Effects on forward rates that result from an induced fit mechanism have been shown
to be a dominant determinant of fidelity in tRNA selection. A body of experimental data
supports this idea by showing that miscoding increases when the differences in GTPase
activation and accommodation rates are decreased either by introduction of a mutation in the
tRNA body or by addition of antibiotics like paromomycin and streptomycin.
We have discussed two general mechanisms used to maintain the high fidelity of protein
synthesis (as well as of DNA replication and transcription). The first mechanism is comprised
of editing and kinetic proofreading. Although different in detail, both strategies amplify the
available discrimination power, determined by differences in free energy of binding, by having
more than one selective step. Of these two strategies, editing has an advantage arising from the
use of two distinct sites that scrutinize different properties of the substrate. The second
mechanism, induced fit, depends on substrate-specific conformational changes that result in
selective modulation of forward rate constants, permitting high fidelity discrimination when
rapid rates are essential. Such distinct solutions for different enzymes ultimately result from
the evolutionary constraints imposed by the diverse requirements for fidelity, speed and
efficiency on each molecular problem.

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