Gene

Expression
TRANSLATION
Transcription to Translation

 The information for the proteins found in a cell is

encoded in genes of the genome of the cell.
 A protein coding gene is expressed by transcription
of the gene to produce an mRNA followed by
translation of the mRNA.
 Translation involves the conversion of the base
sequence of the mRNA into the amino acid
sequence of a polypeptide.
 The base sequence information that specifies the
amino acid sequence of a polypeptide is called the
genetic code.
 Here we learn about how the nucleotide sequence
of mRNA is translated into the amino acid sequence
of a polypeptide
Translation

 A protein consists of one or more molecular

subunits called polypeptides, which are
themselves composed of smaller building blocks,
the amino acids, linked together by peptide
bonds to form long chains.
 The primary amino acid sequence of a protein
determines its secondary, tertiary, and quaternary
structure and hence its functional state.
The Nature of the Genetic
Code
The genetic code is a triplet code : A set of three
nucleotides (a codon) in mRNA code to form one
amino acid in a polypeptide chain.
Characteristics of the Genetic
Code

1) The code is a triplet code.

2) The code is comma free; that is, it is continuous.
3) The code is non-overlapping.
4) The code is almost universal.
5) The code is “degenerate.”
6) The code has start and stop signals.
7) Wobble occurs in the anticodon.
Genetic code is a triplet code
1. The code is a triplet code.

 Each mRNA codon that specifies an

amino acid in a polypeptide chain
consists of three nucleotides.
2. The code is comma free; that is, it is
continuous.
 The mRNA is read continuously, three nucleotides
at a time, without skipping any nucleotides of the
message.
3. The code is non-overlapping

 The mRNA is read in successive groups of

three nucleotides.
4. The code is almost universal.
 Almost all organisms share the same genetic
language.
5. The code is degenerate.
 More than one codon occurs for each amino acid.
 The exceptions are AUG, which alone codes for methionine, and
UGG, which alone codes for tryptophan.
 This multiple coding is called the degeneracy or redundancy of the
code.
6. The code has start and stop signals.
 Specific start and stop signals for protein synthesis are
contained in the code.
 Only 61 of the 64 codons specify amino acids.
 These codons are called sense codons.
 The other three codons—UAG (amber), UAA (ochre),
and UGA (opal)—do not specify an amino acid.
 These three codons are the stop codons, also called
nonsense codons or chain-terminating codons.
7. Wobble occurs in the anticodon.

 Since 61 sense codons specify amino acids in mRNA, a

total of 61 tRNA molecules could have the appropriate
anticodons.
 According to the wobble hypothesis proposed by Francis
Crick, the complete set of 61 sense codons can be read
by fewer than 61 distinct tRNAs.
 Specifically, the base at the 5’ end of the anticodon
complementary to the base at the 3’ end of the codon—
the third letter—is not as constrained three dimensionally as
the other two bases.
 As a result, less exact base pairing can occur: the base at
the 5’ end of the anticodon can pair with more than one
type of base at the 3’ end of the codon—
 In other words, the 5’-base of the anticodon can wobble.
Wobble Hypothesis
Genetic Code

 The genetic code is a triplet code in which each

codon (a set of three contiguous bases) in an
mRNA specifies one amino acid.
 The code is degenerate: some amino acids are
specified by more than one codon.
 The genetic code is non-overlapping and almost
universal.
 Specific codons are used to signify the start and
end of protein synthesis.
Translation:
The Process of Protein Synthesis

 Polypeptide synthesis takes place on ribosomes, where

the genetic message encoded in mRNA is translated.
 The mRNA is translated in the 5’-to-3’ direction.
 The polypeptide is made in the N-terminal–to–C-terminal
direction.
 Amino acids are brought to the ribosome bound to tRNA
molecules.
 Transfer RNA

During translation of mRNA, each transfer

RNA (tRNA) brings a specific amino acid to
the ribosome to be added to a growing
polypeptide chain. The correct amino acid
sequence of a polypeptide is achieved as a
result of:
(1) the binding of each amino acid to a
specific tRNA; and
(2) the binding between the codon of the
mRNA and the complementary anticodon in
the tRNA.
Structure of tRNA.
 Structure of tRNA.
 tRNAs are 75 to 90 nucleotides long, each type having a
different sequence.
 The nucleotide sequences of all tRNAs can be arranged into a
cloverleaf structure.
 The cloverleaf results from complementary base pairing
between different sections of the molecule, producing four base
paired “stems” separated by four loops: I, II, III, and IV.
 Loop II contains the three-nucleotide anticodon sequence,
which pairs with a three-nucleotide codon sequence in mRNA
by complementary base pairing during translation.
 This codon–anticodon pairing is crucial to the addition of the
amino acid specified by the mRNA to the growing polypeptide
chain.
 All tRNA molecules have the sequence 5’-CCA-3’ at their 3’
ends.
 All tRNA molecules also have a number of bases modified
chemically by enzyme reactions, with different arrays of
modifications on each tRNA type
Recognition of the tRNA Anticodon by the
mRNA Codon.

 The mRNA codon recognizes the tRNA anticodon, and not the
amino acid carried by the tRNA.
 This was proved by G. von Ehrenstein, B. Weisblum, and S.
Benzer.
Adding an Amino Acid to tRNA

 The correct amino acid is attached to the tRNA

by an enzyme called aminoacyl–tRNA synthetase.
 The process is called aminoacylation, or charging,
and produces an aminoacyl–tRNA (or charged
tRNA).
 Aminoacylation uses energy from ATP hydrolysis.
 There are 20 different aminoacyl–tRNA
synthetases, one for each of the 20 different
amino acids.
 Each enzyme recognizes particular structural
features of the tRNA or tRNAs it aminoacylates.
Adding an Amino Acid to tRNA
 Adding an Amino Acid to tRNA
 Figure shows the charging of a tRNA molecule to produce valine–
tRNA (Val–tRNA).
 First, the amino acid and ATP bind to the specific aminoacyl–tRNA
synthetase enzyme.
 The enzyme then catalyzes a reaction in which the ATP is hydrolyzed
to AMP, which joins to the amino acid as AMP to form aminoacyl–
AMP.
 Next, the tRNA molecule binds to the enzyme, which transfers the
amino acid from the aminoacyl–AMP to the tRNA and displaces the
AMP.
 The enzyme then releases the aminoacyl–tRNA molecule.
 Chemically, the amino acid attaches at the 3’ end of the tRNA by a
covalent linkage between the carboxyl group of the amino acid and
the 3’OH or 2’OH group of the ribose of the adenine nucleotide
found at the 3’ end of every tRNA.
Attachment of an amino acid to a
tRNA molecule
 Ribosomal RNA and Ribosomes

 Polypeptide synthesis takes place on ribosomes, many thousands of

which occur in each cell.
 Ribosomes bind to mRNA and facilitate the binding of the tRNA to
the mRNA so that a polypeptide chain can be synthesized.
 In both prokaryotes and eukaryotes, ribosomes consist of two
unequally sized subunits—the large and small ribosomal subunits—
each of which consists of a complex between RNA molecules and
proteins.
 Each subunit contains one or more rRNA molecules and a large
number of ribosomal proteins.
Ribosomal RNA and Ribosomes

 The bacterial ribosome has a size of 70S and consists of two

subunits of sizes 50S (large subunit) and 30S (small subunit).
 Eukaryotic ribosomes are larger and more complex than their
prokaryotic counterparts, and they vary in size and
composition among eukaryotic organisms.
 Mammalian ribosomes, for example, have a size of 80S and
consist of a large 60S subunit and a small 40S subunit.
 Each ribosomal subunit contains one or more specific rRNA
molecules and a number of ribosomal proteins.
 Bacterial ribosomes contain three rRNA molecules—the 23S
rRNA and 5S rRNA in the large subunit, and the 16S rRNA in the
small subunit.
 Eukaryotic ribosomes contain four rRNA molecules—the 28S
rRNA, 5.8S rRNA, and 5S rRNA in the large subunit, and the 18S
rRNA in the small subunit.
 The rRNA molecules play a structural role in ribosome and
have a functional role in several steps of translation.
Initiation of Translation

 The three basic stages of protein synthesis—

initiation, elongation, and termination—are similar
in bacteria and eukaryotes.
 Initiation involves an mRNA molecule, a ribosome,
a specific initiator tRNA, protein initiation factors
(IF), and GTP (guanosine triphosphate).
Initiation of Translation
 In bacteria, the first step in the initiation of translation is the interaction of the
30S (small) ribosomal subunit to which IF-1 and IF-3 are bound with the region
of the mRNA containing the AUG initiation codon .
 IF-3 aids in the binding of the subunit to mRNA and prevents binding of the 50S
ribosomal subunit to the 30S subunit.
 The AUG initiation codon alone is not sufficient to indicate where the 30S
subunit should bind to the mRNA.
 A sequence upstream (to the 5’ side in the leader of the mRNA) of the AUG
called the ribosome-binding site (RBS) is also needed.
 In the 1970s, John Shine and Lynn Dalgarno hypothesized that the purine-rich
RBS sequence (5’-AGGAG-3’ or some similar sequence) and sometimes other
nucleotides in this region could pair with a complementary pyrimidine-rich
region (always containing the sequence 5’-UCCUCC-3’) at the 3’ end of 16S
rRNA ).
 Joan Steitz was the first to demonstrate this pairing experimentally.
 The mRNA RBS region is now commonly known as the Shine–Dalgarno
sequence.
 Most of the RBSs are 8 to 12 nucleotides upstream from the initiation codon
 The model is that the formation of complementary base pairs between the
mRNA and 16S rRNA allows the small ribosomal subunit to locate the true
Initiation of Translation contd…

 The next step in the initiation of translation is the binding of a special

initiator tRNA to the AUG start codon to which the 30S subunit is bound.
 In both prokaryotes and eukaryotes, the AUG initiator codon specifies
methionine.
 As a result, newly made proteins in both types of organisms begin with
methionine. In many cases, the methionine is removed later.
 In bacteria, the initiator tRNA is tRNA.fMet, which has the anticodon 5’-
CAU-3’ to bind to the AUG start codon.
 This tRNA carries a modified form of methionine formylmethionine
(fMet), in which a formyl group has been added to the amino group of
methionine.
 That is, first, methionyl–tRNA synthetase catalyzes the addition of
methionine to the tRNA.
 Then the enzyme transformylase adds the formyl group to the
methionine.
 The resulting molecule is designated fMet–tRNA.fMet
Initiation of Translation contd…
 When an AUG codon in an mRNA molecule is encountered at a position other
than the start of the amino acid-coding sequence, a different tRNA, called
tRNA.Met, is used to insert methionine at that point in the polypeptide chain.
 This tRNA is charged by the same aminoacyl–tRNA synthetase as is tRNA.fMet
to produce Met–tRNA.Met.
 However, tRNA.Met and tRNA.fMet molecules are coded for by different
genes and have different sequences.
 The initiator tRNA, fMet–tRNA.fMet, is brought to the 30S subunit–mRNA
complex by IF-2, which also carries a molecule of GTP.
 The initiator tRNA binds to the subunit in the P site but subsequently, all
aminoacyl–tRNAs that come to the ribosome bind to the A site.
 However, IF-1 bound to the 30S subunit is blocking the A site so that only the P
site is available for the initiator tRNA to bind to.
 Formed at this point is the 30S initiation complex, consisting of the mRNA, 30S
subunit,
 initiator tRNA, and the initiation factors.
 Next, the 50S ribosomal subunit binds, leading to GTP hydrolysis and the
release of the three initiation factors.
 The final complex is called the 70S initiation complex.
Initiation of protein synthesis in bacteria
Initiation of Translation in Eukaryotes
 The initiation of translation is similar in eukaryotes, although the process is more complex
and involves many more initiation factors, called eukaryotic initiation factors (eIF), than is
the case in bacteria.
 The main differences are that:
 (1) the initiator methionine is unmodified, although a special initiator tRNA still brings it to
the ribosome; and
 (2) Shine–Dalgarno sequences are not found in eukaryotic mRNAs. Instead, the eukaryotic
ribosome uses another way to find the AUG initiation codon.
 First, a eukaryotic initiator factor eIF-4F— a multimer of several proteins, including eIF-4E,
the cap-binding protein (CBP)—binds to the cap at the 5’ end of the mRNA.
 Then, a complex of the 40S ribosomal subunit with the initiator Met–tRNA, several eIF
proteins, and GTP binds, together with other eIFs, and moves along the mRNA, scanning
for the initiator AUG codon.
 The AUG codon is embedded in a short sequence—called the Kozak sequence, after
Marilyn Kozak—which indicates that it is the initiator codon.
 This process is called the scanning model for initiation. The AUG codon is almost always the
first AUG codon from the 5’ end of the mRNA; but, to be an initiator codon, it must be in
an appropriate sequence context.
 Once the 40S subunit finds this AUG, it binds to it, and then the 60S ribosomal subunit binds,
displacing the eIFs (except for eIF-4F, which is needed for the subsequent initiation of
translation), producing the 80S initiation complex with the initiator Met–tRNA bound to the
mRNA in the P site of the ribosome.
Initiation of Translation in Eukaryotes
Elongation of the Polypeptide Chain

 After initiation is complete, the next stage is

elongation.
 This phase has three steps:
1. Aminoacyl–tRNA (charged tRNA) binds to the
ribosome in the A site.
2. A peptide bond forms.
3. The ribosome moves (translocates) along the
mRNA one codon.
 As with initiation, elongation requires accessory
protein factors, here called elongation factors
(EF), and GTP.
 Elongation is similar in eukaryotes.
Elongation of the Polypeptide Chain
Binding of Aminoacyl–tRNA.

 At the start of elongation, the anticodon of fMet–tRNA is hydrogen

bonded to the AUG initiation codon in the P site of the ribosome.
 The next codon in the mRNA is in the A site.
 Next, the appropriate aminoacyl–tRNA binds to the codon in the A site.
 This aminoacyl–tRNA is brought to the ribosome bound to EF-Tu–GTP, a
complex of the protein elongation factor EF-Tu and a molecule of GTP.
 When the aminoacyltRNA binds to the codon in the A site, GTP
hydrolysis releases EF-Tu–GDP.
 EFTu is recycled.
 First, a second elongation factor, EF-Ts, binds to EF-Tu and displaces the
GDP.
 Next, GTP binds to the EF-Tu–EF-Ts complex to produce an EF-Tu–GTP
complex simultaneously with the release of EF-Ts.
 An aminoacyl-tRNA binds to the EF-Tu–GTP, and that complex can bind
to the A site in a ribosome when the complementary codon is exposed.
 The process is highly similar in eukaryotes, with eEF-1A playing the role of
EF-Tu, and eEF-1B playing the role of EF-Ts.
Peptide Bond Formation

 The ribosome maintains the two aminoacyl–tRNAs in the P and

A sites in the correct positions, so that a peptide bond can
form between the two amino acids.
 Two steps are involved in the formation of this peptide bond.
 First, the bond between the amino acid and the tRNA in the P
site is cleaved. In this case, the breakage is between the fMet
and its tRNA.
 Second, the peptide bond is formed between the now-freed
fMet and the aminoacid attached to the tRNA in the A site in
a reaction catalyzed by peptidyl transferase.
 Once the peptide bond has formed, a tRNA without an
attached amino acid (an uncharged tRNA) is left in the P site.
 The tRNA in the A site, now called peptidyl–tRNA, has the first
two aminoacids of the polypeptide chain attached to it.
Translocation

 In the last step in the elongation cycle, translocation,

the ribosome moves one codon along the mRNA
toward the 3’ end.
 In bacteria, translocation requires the activity of
another protein elongation factor, EF-G.
 An EF-G–GTP complex binds to the ribosome, GTP is
hydrolyzed, and translocation of the ribosome
occurs along with displacement of the uncharged
tRNA away from the P site.
 It is possible that GTP hydrolysis changes the structure
of EF-G, which facilitates the translocation event.
 Translocation is similar in eukaryotes; the elongation
factor in this case is eEF-2, which functions like
bacterial EF-G.
Translocation contd….

 The uncharged tRNA moves from the P site and then binds transiently to
the E site in the 50S ribosomal subunit, blocking the next aminoacyl–
tRNA from binding to the A site until translocation is complete and the
peptidyl–tRNA is bound correctly in the P site.
 Once that has occurred, the uncharged tRNA is then released from the
ribosome.
 After translocation, EF-G is released and then reused.
 During the translocation step, the peptidyl–tRNA remains attached to its
codon on the mRNA.
 The ribosome moves and the peptidyl–tRNA is now located in the P site
(hence the name peptidyl site).
 After the completion of translocation, the A site is vacant.
 An aminoacyl–tRNA with the correct anticodon binds to the newly
exposed codon in the A site, reiterating the process already described.
 The whole process is repeated until translation terminates at a stop
codon.
Translocation contd….

 In both bacteria and eukaryotes, once the ribosome

moves away from the initiation site on the mRNA,
another initiation event occurs.
 The process is repeated until, typically, several
ribosomes are translating each mRNA
simultaneously.
 The complex between an mRNA molecule and all
the ribosomes that are translating it simultaneously is
called a polyribosome, or polysome.
 Each ribosome in a polysome translates the entire
mRNA and produces a single, complete
polypeptide.
 Polyribosomes enable a large number of
polypeptides to be produced quickly and efficiently
from a single mRNA.
Termination of Translation

 The termination of translation is signaled by one of three stop

codons (UAG, UAA, and UGA), which are the same in
prokaryotes and eukaryotes.
 The stop codons do not code for any amino acid, so no tRNAs
in the cell have anticodons for them.
 The ribosome recognizes a stop codon with the help of
proteins called release factors (RF), which have shapes
mimicking that of a tRNA including regions that read the
codons and then initiate a series of specific termination
events.
 In E. coli, there are three RFs, two of which read the stop
codons: RF1 recognizes UAA and UAG, and RF2 recognizes
UAA and UGA.
 The binding of RF1 or RF2 to a stop codon triggers peptidyl
transferase to cleave the polypeptide from the tRNA in the P
site.
 The polypeptide then leaves the ribosome.
Termination of Translation

 Next, RF3–GDP binds to the ribosome, stimulating the release of the RF from
the stop codon and the ribosome.
 GTP now replaces the GDP on RF3, and RF3 hydrolyses the GTP, which allows
RF3 to be released from the ribosome.
 An additional important step is the deconstruction of the remaining complex
of ribosomal subunits, mRNA, and uncharged tRNA so that the ribosome and
tRNA may be recycled.
 In E. coli, ribosome recycling factor (RRF)—the shape of which mimics that of
a tRNA— binds to the A site.
 Then EF-G binds, causing translocation of the ribosome and thereby moving
RRF to the P site and the uncharged tRNA to the E site.
 The RRF releases the uncharged tRNA, and EF-G releases RRF, causing the
two ribosomal subunits to dissociate from the mRNA.
 In eukaryotes, the termination process is similar to that in bacteria.
 In this case, a single release factor— eukaryotic release factor 1 (eRF1)—
recognizes all three stop codons, and eRF3 stimulates the termination events.
 Ribosome recycling occurs in eukaryotes, but there is no equivalent of RRF.
Protein Sorting in the Cell
 In bacteria and eukaryotes, some proteins may be secreted; and in
eukaryotes, some other proteins must be placed in different cell
compartments, such as the nucleus, mitochondrion, chloroplast,
lysosome.
 The sorting of proteins to their appropriate compartments is under
genetic control, in that specific “signal” or “leader” sequences on the
proteins direct them to the correct organelles.
 In an eukaryotic cell such proteins are passaged through the
endoplasmic reticulum (ER) and Golgi apparatus.
 In 1975, Gunther Blobel, B. Dobberstein, and colleagues found that
secreted proteins and other proteins sorted by the Golgi initially
contain extra amino acids at the amino terminal end.
 Blobel’s work led to the signal hypothesis, which states that proteins
sorted by the Golgi bind to the ER by a hydrophobic amino terminal
extension (the signal sequence) to the membrane that is
subsequently removed and degraded.
 Blobel won the Nobel Prize in Physiology or Medicine in 1999 for this
work.
 Protein Sorting in the Cell
 The signal sequence of a protein destined for the ER consists of about 15 to 30
N-terminal amino acids.
 When the signal sequence is produced by translation and exposed on the
ribosome surface, a cytoplasmic signal recognition particle (SRP, an RNA–
protein complex) binds to the sequence and blocks further translation of the
mRNA until the growing polypeptide–SRP–ribosome–mRNA complex reaches
and binds to the ER.
 The SRP binds to an SRP receptor in the ER membrane, causing the firm binding
of the ribosome to the ER with release of the SRP, and the resumption of
translation.
 The growing polypeptide extends through the ER membrane into the cisternal
space of the ER.
 Once the signal sequence is fully into the cisternal space of the ER, it is
removed from the polypeptide by the enzyme signal peptidase.
 When the complete polypeptide is entirely within the ER cisternal space, it is
typically modified by the addition of specific carbohydrate groups to produce
glycoproteins. The glycoproteins are then transferred in vesicles to the Golgi
apparatus, where most of the sorting occurs.
 Proteins destined to be secreted, for example, are packaged into secretory
storage vesicles, which migrate to the cell surface, where they fuse with the
plasma membrane and release their packaged proteins to the outside of the
cell.
Post-translational Processing of Proteins
 Translation is not the end of the genome expression pathway.
 The polypeptide that emerges from the ribosome is inactive,
and before taking on its functional role in the cell must undergo
at least the first of the following four types of post-translational
processing:
• Protein folding. The polypeptide is inactive until it is folded into its
correct tertiary structure.
• Proteolytic cleavage. Some proteins are processed by cutting
events carried out by enzymes called proteases. These cutting
events may remove segments from one or both ends of the
polypeptide, resulting in a shortened form of the protein, or they
may cut the polypeptide into a number of different segments, all
or some of which are active.
• Chemical modification. Individual amino acids in the
polypeptide might be modified by attachment of new chemical
groups.
• Intein splicing. Inteins are intervening sequences in some
proteins, similar in a way to introns in mRNAs. They have to be
removed and the exteins ligated in order for the protein to
become active.
Protein folding is aided by molecular
chaperones
 Most of our current understanding of protein folding in the
cell is founded on the discovery of proteins that help other
proteins to fold.
 These are called molecular chaperones and have been
studied in detail in E. coli. Both eukaryotes and archaea
possess equivalent proteins, although some of the details of
the way they work are different.
 The molecular chaperones in E. coli can be divided into
two groups:
• The Hsp70 chaperones, which include the proteins
called Hsp70 (coded by the dnaK gene and sometimes
called DnaK protein), Hsp40 (coded by dnaJ) and GrpE;
• The chaperonins, the main version of which in E. coli is
the GroEL/GroES complex.
Processing by proteolytic cleavage
Proteolytic cleavage has two functions in post-translational
processing of proteins:
• It is used to remove short pieces from the N- and/or C-
terminal regions of polypeptides, leaving a single shortened
molecule that folds into the active protein.
• It is used to cut polyproteins into segments, all or some of
which are active proteins.

These events are relatively common in eukaryotes but less

frequent in bacteria.
Processing by chemical modification
 The genome has the capacity to code for 21 different amino
acids: the 20 specified by the standard genetic code, and
selenocysteine, which is inserted into polypeptides by the
context-dependent reading of a 5 -UGA-3 codon.
 This range is increased by post-translational chemical
modification of proteins, which results in a vast variety of
different amino acid types.
 The simpler types of modification occur in all organisms; the
more complex ones, especially glycosylation, are rare in
bacteria.
 The simplest types of chemical modification involve addition of
a small chemical group (e.g. an acetyl, methyl or phosphate
group) to an amino acid side chain, or to the amino or
carboxyl groups of the terminal amino acids in a polypeptide.
 Over 150 different modified amino acids have been
documented in different proteins, with each modification
carried out in a highly specific manner, the same amino acids
being modified in the same way in every copy of the protein.
Examples of post-translational
chemical modifications

 Addition of small chemical groups

Acetylation, Methylation, Phosphorylation

 Addition of sugar side chains

O-linked and N-linked glycosylation

 Addition of lipid side chains

Acylation, N-myristoylation

 Addition of biotin
Biotinylation
 Inteins
 The final type of post-translational processing is intein
splicing, a protein version of the more extensive intron
splicing that occurs with pre-RNAs.
 Inteins are internal segments of proteins that are
removed soon after translation, the two external
segments or exteins becoming linked together.
 Most inteins are approximately 150 amino acids in length
and, like pre-mRNA introns, the sequences at the splice
junctions of inteins have some similarity.
 In particular, the first amino acid of the downstream
extein is cysteine, serine or threonine. A few other amino
acids within the intein sequence are also conserved.
 These conserved amino acids are involved in the splicing
process, which is self-catalyzed by the intein itself.