BCH 303: NUCLEIC ACIDS

BY

PROF. E. T. OLAYINKA

1|Page
Genetic code
Genetic code is the sequence of nucleotides in deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA) that determines the amino acid sequence of proteins. Though the linear sequence
of nucleotides in DNA contains the information for protein sequences, proteins are not made
directly from DNA. Instead, a messenger RNA (mRNA) molecule is synthesized from the DNA
and directs the formation of the protein.
RNA is composed of four nucleotides: adenine (A), guanine (G), cytosine (C), and uracil (U).
Three adjacent nucleotides constitute a unit known as the codon, which codes for an amino
acid. For example, the sequence AUG is a codon that specifies the amino acid methionine.
There are 64 possible codons, three of which do not code for amino acids but indicate the
end of a protein. The remaining 61 codons specify the 20 amino acids that make up proteins.
The AUG codon, in addition to coding for methionine, is found at the beginning of every mRNA
and indicates the start of a protein. Methionine and tryptophan are the only two amino acids
that are coded for by just a single codon (AUG and UGG, respectively). The other 18 amino
acids are coded for by two to six codons. Because most of the 20 amino acids are coded for
by more than one codon, the code is called degenerate.
Characteristic of Genetic Code

• The genetic code is the set of rules by which a linear sequence of nucleotides specifies
the linear sequence of a polypeptide.
• That is, they specify how the nucleotide sequence of an mRNA is translated into the
amino acid sequence of a polypeptide.
• Thus, the relationship between the nucleotide sequence of the mRNA and the amino
acid sequence of the polypeptide is the genetic code.
• The nucleotide sequence is read as triplets called codons.
Principles of the Genetic Code
• The genetic code consists of 64 different codons, each of which codes for 1 of the 20
amino acids.
• A codon consists of a triplet of nucleotide bases.

Characteristics of the Genetic Code

The genetic code is endowed with many characteristic properties which have actually been
proved by definite experimental evidences.

2|Page
 1. Triplet nature:
• Singlet and doublet codes are not adequate to code for 20 amino acids; therefore, it
was pointed out that triplet code is the minimum required.
2. Degeneracy
• The code is degenerate which means that the same amino acid is coded by more than
one base triplet.
• Degeneracy does not imply lack of specificity in protein synthesis.
• It merely means that a particular amino acid can be directed to its place in the peptide
chain by more than one base triplets.
• For example, the three amino acids arginine, alanine and leucine each have six
synonymous codons.
• The code degeneracy is basically of 2 types: partial and complete.
• In partial degeneracy, the first two nucleotides are identical but the third (i.e., 3′ base)
nucleotide of the degenerate codon differs; for example, CUU and CUC code for
leucine.
• Complete degeneracy occurs when any of the 4 bases can take third position and still
code for the same amino acid; for example, UCU, UCC, UCA and UCG all code for serine.

3|Page
 3. Non-overlapping
• The genetic code is nonoverlapping, i.e.,the adjacent codons do not overlap.
• A nonoverlapping code means that the same letter is not used for two different
codons. In other words, no single base can take part in the formation of more than one
codon.
4. Commaless
• The genetic code is commaless (or comma-free). There is no signal to indicate the end
of one codon and the beginning of the next.
• There are no intermediary nucleotides (or commas) between the codons.
5. Non-ambiguity
• Non-ambiguous code means that there is no ambiguity about a particular codon.
• A particular codon will always code for the same amino acid.
• While the same amino acid can be coded by more than one codon (the code is
degenerate), the same codon shall not code for two or more different amino acids
(non-ambiguous).
6. Universality
• Universality of the code means that the same sequences of 3 bases encode the same
amino acids in all life forms from simple microorganisms to complex, multicelled
organisms such as human beings.
• Although the code is based on work conducted on the bacterium Escherichia coli but
it is valid for other organisms.
• The genetic code applies to all modern organisms with only minor exceptions, such as
the yeast, mitochondria, and the Mycoplasma.
7. Polarity
• The genetic code has polarity, that is, the code is always read in a fixed direction, i.e.,
in the 5′ → 3′ direction.
• It is apparent that if the code is read in opposite direction (i.e., 3′ → 5′), it would specify
2 different proteins, since the codon would have reversed base sequence.
Special Codons
A. Chain Initiation Codons
• The triplets AUG and GUG play double roles in E. coli.
• When they occur in between the two ends of a cistron (intermediate position), they
code for the amino acids methionine and valine, respectively in an intermediate
position in the protein molecule.

4|Page
 • But when they occur immediately after a terminator codon, they act as “chain
initiation” (C.I.) signals or “starter codons” for the synthesis of a polypeptide chain.
B. Chain Termination Codons
• The 3 triplets UAA, UAG, UGA do not code for any amino acid.
• When any one of them occurs immediately before the triplet AUG or GUG, it causes
the release of the polypeptide chain from the ribosome.
• They are also called as stop codons.
• They are also called chain termination codons because these codons are used by the
cell to signal the natural end of translation of a particular peptidyl chain. However,
their inclusion in any mRNA results in the abrupt termination of the message at the
point of their location even though the polypeptide chain has not been completed.
C. Sense Codons
61 codons, which code for particular amino acids are termed as sense codons.
D. Non-Sense Codons
• Triplets UAA, UAG, UGA do not code for any amino acid.
• They were originally described as non-sense codons.
• However, the so-called non-sense codons have now been found to be of “special
sense”.
• These special-sense codons perform the function of punctuating genetic message like
a full stop at the end of a sentence.

Reading frame

• As shown in Figure above, the codon AUG codes for the amino acid methionine. This
codon is also the start codon that begins translation.
• The start codon establishes the reading frame of mRNA. The reading frame is the way
the letters are divided into codons.
• After the AUG start codon, the next three letters are read as the second codon. The
next three letters after that are read as the third codon, and so on. This is illustrated
in Figure below.
• The mRNA molecule is read, codon by codon, until a stop codon is reached. UAG, UGA,
and UAA are all stop codons. They do not code for any amino acids. Stop codons are
also known as termination codons.

5|Page
 • To reliably get from an mRNA to a protein, we need one more concept: that of reading
frame. Reading frame determines how the mRNA sequence is divided up into codons
during translation.
• That's a pretty abstract concept, so let's look at an example to understand it better.
The mRNA below can encode three totally different proteins, depending on the frame
in which it's read:

6|Page
So, how does a cell know which of this protein to make? The start codon is the key signal.
Because translation begins at the start codon and continues in successive groups of three, the
position of the start codon ensures that the mRNA is read in the correct frame (in the example
above, in Frame 3).
Mutations (changes in DNA) that insert or delete one or two nucleotides can change the
reading frame, causing an incorrect protein to be produced "downstream" of the mutation
site:

7|Page
Transfer RNA (tRNA)- Definition, Structure, Processing, Types, Functions
Definition of tRNA- Transfer RNA or tRNA is a type of RNA molecule that helps to decode
information present in mRNA sequences into specific proteins.
• tRNA molecule is a carrier of amino acid that brings appropriate amino acid to
ribosome based on the codon present in mRNA sequence.
• tRNA is also known as an adaptor molecule as it translates the codons present in
mRNA sequences into amino acids.
• tRNA is only 70-90 nucleotides in length, making it the smallest out of the three main
RNAs (mRNA and rRNA).
• The molecular weight of the tRNA molecule ranges from 25,000 to 30,000 Dalton.
• tRNA is encoded by DNA in the cell nucleus and transcribed with the help of RNA
polymerase ΙΙΙ.

Properties of tRNA
• tRNA can make hydrogen bonds with mRNA and also can form ester linkage with amino
acids, thus linking mRNA with the amino acid at the site of protein synthesis.
• tRNA bond complementary with each three-nucleotide of mRNA called codons and
this happens in an antiparallel fashion. The part of tRNA that binds with the codon of
mRNA is termed as anticodon arm of tRNA.
• This complementary base pairing of tRNA and mRNA makes the correct translation of
the correct amino acid that is inserted into the growing polypeptide chain.

8|Page
 • The tRNA is a single-stranded RNA molecule that has the proximity to fold upon itself
and creates an intra complementary base pairing which gives raise to hydrogen-
bonded stems and associated loops that contains nucleotides with modified bases.
• In addition to the usual bases i.e., Adenine, Uracil, Cytosine, and Guanosine, tRNA also
contains modified unusual bases like for Adenine, there is an unusual base called
Inosine, likewise in place of Uracil, there are Psuedouracil and Dihydrouridine.
• These modified unusual bases and intramolecular base pairing of tRNA make it stable
and protect it from getting degraded by the enzyme RNase.

Kinds of tRNA Structure

The tRNA has three kinds of structure:
1. Primary structure
• A linear single strand of ribonucleotides running in a 5′ to 3′ direction.
• The number of ribonucleotides ranges from 60-90, the most commonly found length
is 76 ribonucleotides.
• A single tRNA molecule having a primary structure consists of about 20% of modified
bases.
• Based on these modified bases, the linear sequence of tRNA molecule can be divided
into three arms: D arm containing a modified base Dihydrouridine, Anticodon arm
containing unusual bases like Inosine derived from Adenine, or Pseudouridine from
Uracil or Lysidine from Cytosine, TΨC arm containing modified base Pseudouridine.
• In addition to these three arms with modified bases, an arm is present at the 3′
terminal usually containing CCA nucleotide and is referred to as Acceptor arm that acts
as an attachment site for incoming amino acids.
• D arm serves as a recognition site for the enzyme aminoacyl tRNA synthetase.
• The Anticodon arm binds with the codon of mRNA during protein synthesis.
• TΨC arms contain a ribosome binding site.
2. Secondary structure (Cloverleaf model)
• The secondary structure of tRNA is of 74-95 nucleotides sequences that are stable and
fold on itself to give a clover leaf-like structure containing four arms sometimes even
five in longer tRNAs.
• These four base-paired arms are namely, Acceptor arm, D-arm, TΨC arm, and
Anticodon arm.
• The acceptor’s arm is a 7 to 9 base pair in length, this arm is formed by base pairing of
5′ terminal and 3′ terminal nucleotides.

9|Page
 • Out of these four base-paired arms, three arms end up in a loop due to unpaired base-
pairing forming D- loop, Anticodon loop, and TΨC loop respectively. However, the
acceptor arm does not form a loop but contains an extension of the free 3′ hydroxyl
group comprising of CCA nucleotides that are used to attach amino acids.
• The carboxylic group of amino acids joins with free 3′ hydroxyl of Adenine in CCA to
aminoacyl tRNA, the process is also referred to as charging of tRNA and is catalyzed by
an enzyme, aminoacyl tRNA synthetase.
• The acceptor arm is located opposite to Anticodon arm.
• D arm consists of a stem that is about 4 to 5 base pairs in length which end in a loop
that usually contains modified pyrimidine base nucleotides, Dihydrouridine.
• D arm serves as a recognition site for the enzyme aminoacyl tRNA synthetase.
• Anticodon arm consists of a stem that is about 6 base pairs in length which end in a
loop due to 7 unpaired bases. Out of which three bases form an anticodon.
• This first anticodon base is sometimes modified with unusual bases like Inosine derived
from Adenine, or Pseudouridine from Uracil, or Lysidine from Cytosine.
• This anticodon base of the anticodon arm recognizes codon in mRNA and binds to it
during protein synthesis.
• The TΨC arm consists of a stem that is 5 base pairs long which ends in a loop that
further contains 7 unpaired base pairs, usually containing Thymine, Pseudouridine and
Cytosine hence the name TΨC arm.
• The TΨC loop contains a ribosome binding site.
• In addition to four base-paired arms, a variable arm can usually be present in long
tRNAs. These variable arms vary in length from 3-21 nucleotides depending on the
type of amino acid they code.
• The variable arm lies between the anticodon arm and TΨC arm, and is usually short
and looks sort of different from the regular four arms. The base pairing is rarely seen
in this arm, so it appears as loops due to unpaired bases.
• The variable arm accounts for the stability of a tRNA molecule.
3. Tertiary structure
• It is a three-dimensional structure, where tRNA adopts an L-shaped structure.
• This L-shape is formed when the stem of acceptor and TΨC arms stack at one side and
stem of anticodon and D arms stack on another side and both forms extended helices.
• Both of these extended helices align at a right angle, and in doing so, the D-loop and
TΨC loop align together.

10 | P a g e
 • The tertiary structure is the most stable structure out of all, and this stability is
acquired due to hydrogen bonding in between nitrogenous bases and also between
nitrogenous bases and the ribose-phosphate backbone.
• To synthesize proteins by reading codon in mRNA a tRNA molecule must have a tertiary
structure.
Types of tRNA
• The types of tRNA are categorized on the types of amino acids it carries. A human
typically has 20 essential amino acids which thus give rise to 20 different types of
tRNA.
• However, alternatively, they can also be categorized based on their anticodons.
• When genes are expressed to proteins. DNA is transcribed to mRNA which is then
translated into protein by tRNA. DNA contains four nucleotides so their possible
combination gives rise to 64 codons.
• Out of 64 codons, 3 codons are stop codons i.e., UAG, UAA, and UGA. These three
codons trigger the termination of translation. In addition to tRNA molecules being
needed to pair with each amino acid. Some other tRNA molecules are also needed to
pair up with these stop codons to halt the protein synthesis.
• Some codons code for the same amino acid, thus the real number of tRNA is not 64.
This redundancy in genetic code is referred to as “wobble”.
• Due to this redundancy, very few species of organisms have exactly 61 codons that
code for amino acids.
• tRNA interacts with codons via its anticodon loop. Base pairing between anticodon
and codon ensures the specificity of amino acids to be incorporated in polypeptide
chains.

Anticodon

• An anticodon is a unit of three nucleotides corresponding to the three bases of an

mRNA codon.
• Each tRNA has a distinct anticodon triplet sequence that can form 3 complementary
base pairs to one or more codons for an amino acid.
• Some anticodons pair with more than one codon due to wobble base pairing.
Frequently, the first nucleotide of the anticodon is one not found on mRNA: inosine,
which can hydrogen bond to more than one base in the corresponding codon position.
• In genetic code, it is common for a single amino acid to be specified by all four third-
position possibilities, or at least by both pyrimidines and purines; for example, the
amino acid glycine is coded for by the codon sequences GGU, GGC, GGA, and GGG.
Other modified nucleotides may also appear at the first anticodon position—
sometimes known as the "wobble position"—resulting in subtle changes to the genetic
code, as for example in mitochondria.
• The possibility of wobble bases reduces the number of tRNA types required: instead
of 61 types with one for each sense codon of the standard genetic code),
• only 31 tRNAs are required to translate, unambiguously, all 61 sense codons.

11 | P a g e
The Wobble Hypothesis: Definition, Statement, Significance
There are more than one codon for one amino acid. This is called degeneracy of genetic code.
To explain the possible cause of degeneracy of codons, in 1966, Francis Crick proposed “the
Wobble hypothesis”.
According to The Wobble Hypothesis, only the first two bases of the codon have a precise
pairing with the bases of the anticodon of tRNA, while the pairing between the third bases of
codon and anticodon may Wobble (wobble means to sway or move unsteadily).
The phenomenon permits a single tRNA to recognize more than one codon. Therefore,
although there are 61 codons for amino acids, the number of tRNA is far less (around 40)
which is due to wobbling.
The Wobble Hypothesis Statement
The wobble hypothesis states that the base at 5′ end of the anticodon is not spatially
confined as the other two bases allowing it to form hydrogen bonds with any of several
bases located at the 3′ end of a codon.
This leads to the following conclusions:
• The first two bases of the codon make normal (canonical) H-bond pairs with the 2nd
and 3rd bases of the anticodon.
• At the remaining position, less stringent rules apply and non-canonical pairing may
occur. The wobble hypothesis thus proposes a more flexible set of base-pairing rules
at the third position of the codon.
• The relaxed base-pairing requirement, or “wobble,” allows the anticodon of a single
form of tRNA to pair with more than one triplet in mRNA.
• The rules: first base U can recognize A or G, first base G can recognize U or C, and first
base I can recognize U, C or A.
Crick’s hypothesis hence predicts that the initial two ribonucleotides of triplet codes are often
more critical than the third member in attracting the correct tRNA.

12 | P a g e
Wobble base pairs

• A wobble base pair is a pairing between two nucleotides in RNA molecules that does
not follow Watson-Crick base pair rules.
• The four main wobble base pairs are guanine-uracil (G-U), hypoxanthine-uracil (I-
U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C).

13 | P a g e
 • In order to maintain consistency of nucleic acid nomenclature, “I” is used for
hypoxanthine because hypoxanthine is the nucleobase of inosine.
• Inosine displays the true qualities of wobble, in that if that is the first nucleotide in the
anticodon then any of three bases in the original codon can be matched with the tRNA.

Ribosomes: Structure, Types and Functions

The ribosome word is derived – ‘ribo’ from ribonucleic acid and ‘somes’ from the Greek word
‘soma’ which means ‘body’.
Ribosomes are tiny spheroidal dense particles (of 150 to 200 A0 diameters) that are primarily
found in most prokaryotic and eukaryotic cells
• They are sites of protein synthesis.
• They are structures containing approximately equal amounts of RNA and proteins and
serve as a scaffold for the ordered interaction of the numerous molecules involved in
protein synthesis.
• The ribosomes occur in cells, both prokaryotic and eukaryotic cells.
• In prokaryotic cells, the ribosomes often occur freely in the cytoplasm.
• In eukaryotic cells, the ribosomes either occur freely in the cytoplasm or remain
attached to the outer surface of the membrane of the endoplasmic reticulum.
• The location of the ribosomes in a cell determines what kind of protein it makes.
• If the ribosomes are floating freely throughout the cell, it will make proteins that will
be utilized within the cell itself.
• When ribosomes are attached to the endoplasmic reticulum, it is referred to as rough
endoplasmic reticulum or rough ER.
• Proteins made on the rough ER are used for usage inside the cell or outside the cell.
• The number of ribosomes in a cell depends on the activity of the cell.
• On average in a mammalian cell, there can be about 10 million ribosomes.

14 | P a g e
Structure of Ribosomes
• A ribosome is made from complexes of RNAs and proteins and is, therefore, a
ribonucleoprotein.
• Around 37 to 62% of RNA is comprised of RNA and the rest is proteins.
• Each ribosome is divided into two subunits:
1. A smaller subunit which binds to a larger subunit and the mRNA pattern, and
2. A larger subunit which binds to the tRNA, the amino acids, and the smaller subunit.
• Prokaryotes have 70S ribosomes respectively subunits comprising the little subunit of
30S and the bigger subunit of 50S.
• Their small subunit has a 16S RNA subunit (consisting of 1540 nucleotides) bound to
21 proteins.
• The large subunit is composed of a 5S RNA subunit (120 nucleotides), a 23S RNA
subunit (2900 nucleotides) and 31 proteins.
• Eukaryotes have 80S ribosomes respectively comprising of little (40S) and substantial
(60S) subunits.
• The smaller 40S ribosomal subunit is prolate ellipsoid in shape and consists of one
molecule of 18S ribosomal RNA (or rRNA) and 30 proteins (named as S1, S2, S3, and
so on).
• The larger 60S ribosomal subunit is round in shape and contains a channel through
which growing polypeptide chain makes its exit.
• It consists of three types of rRNA molecules, i.e., 28S rRNA, 5.8 rRNA and 5S rRNA, and
40 proteins (named as L1, L2, L3 and so on).

15 | P a g e
 • The differences between the ribosomes of bacterial and eukaryotic are used to create
antibiotics that can destroy bacterial infection without harming human cells.
• The ribosomes seen in the chloroplasts of mitochondria of eukaryotes are comprised
of big and little subunits composed of proteins inside a 70S particle.
• The ribosomes share a core structure that is similar to all ribosomes despite
differences in its size.
• The two subunits fit together and work as one to translate the mRNA into a
polypeptide chain during protein synthesis.
• Because they are formed from two subunits of non-equal size, they are slightly longer
in the axis than in diameter.
• During protein synthesis, when multiple ribosomes are attached to the same mRNA
strand, this structure is known as polysome.
• The existence of ribosomes is temporary, after the synthesis of polypeptide the two
sub-units separate and are reused or broken up.
Functions of Ribosomes

• The ribosome is a complex molecular machine, found within all living cells, that serves
as the site of biological protein synthesis (translation).
• Ribosomes link amino acids together in the order specified by messenger RNA (mRNA)
molecules.
• Ribosomes act as catalysts in two extremely important biological processes called
peptidyl transfer and peptidyl hydrolysis.
• The nascent polypeptide chain is protected from the activity of protein digestive
enzymes.
How does the ribosomal movement take place in translation?
• In addition to a binding site for an mRNA molecule, it consists of other 3 binding sites.
for tRNA molecules;
• A site
• P site
• E site
• Amino acid needs to be added to a growing peptide chain.
• The charged tRNA whose base pairs with the complementary codon on the mRNA
molecule enters the A site.
• Then in the new forming polypeptide chain, an amino acid is added which is held by
the tRNA in the adjacent P site.
• Then the large ribosomal subunit moves forward to the E site.

16 | P a g e
 • This process or the cycle is repeated.
• Each time an amino acid is added to the polypeptide chain, where the new protein
grows from its amino to its carboxyl end.
• Finally, the stop codon will be encountered in the mRNA.
• The termination release factors like the RF1 and RF2 recognize the stop codons.
• Then the peptidyl-tRNA bond is hydrolyzed.
• Finally, the newly formed polypeptide is released from the ribosome.

Protein Synthesis: Enzymes, Sites, Steps, Inhibitors

Protein Synthesis is a process of synthesizing proteins in a chain of amino acids known as

polypeptides. It is the second part of the central dogma in genetics.
• It takes place in the ribosomes found in the cytosol or those attached to the rough
endoplasmic reticulum.
• The functions of the ribosome are to read the sequence of the codons in mRNA and
the tRNA molecules that transfer or transport or bring the amino acids to the
ribosomes in the correct sequence. However, other molecules are also involved in the
process of translation such as various enzymatic factors.
• The translation process involves reading the genetic code in mRNA to make proteins.
• The entire translation process can be summarized into three phases: Initiation,
elongation, and termination.

The translation process is guided by machinery composed of:

• Ribosomes
• Transfer RNA (tRNA)
Overview of the Protein Synthesis
• The ribosomal translation is initiated when the ribosomes recognize the starting point
of mRNA, where it binds a molecule of tRNA that bears a single amino acid.
• In prokaryotes, the initial amino acid in N-formylmethionine. during elongation, the
second amino acid is linked to the first one.
• The ribosome then shifts its position on the mRNA and repeats the elongation cycle.
• When the elongation process reaches the stop codon, the amino acid chain folds
spontaneously to form a protein.

17 | P a g e
 • The ribosomes then split into two subunits, but later rejoin before another mRNA is
translated.
• Protein synthesis is facilitated by several catalytic proteins which include initiation,
elongation, termination factors, and guanosine triphosphates (GTP).
• GTP is a molecule that releases energy when converted into guanosine diphosphate
(GDP).
Protein Synthesis Steps /Process in Details
Translation Initiation
• Protein synthesis initiation is triggered by the presence of several initiation factors IF1,
IF2, and IF3, including mRNA, ribosomes, tRNA.
• The small subunit binds to the upstream on the 5′ end at the start of mRNA. The
ribosome scans the mRNA in the 5′ to 3′ direction until it encounters the start codon
(AUG or GUG or UUG). When either of these start codons is present, it is recognized
by the initiator fMet-tRNA (N-formylMet-tRNA). This initiator factor carries the
methionine (Met) which binds to the P site on the ribosome.
• This synthesizes the first amino acid polypeptide known as N-formylmethionine. The
initiator fMet-tRNA has a normal methionine anticodon therefore it inserts the N-
formylmethionine. This means that methionine is the first amino acid that is added
and appears in the chain.
• Generally, there are three steps in the initiation process of translation;
1. Initiation of the binding of mRNA to the small ribosome subunit (the 30S), stimulating
the initiator factor IF3. this dissociates the ribosomal subunits into two.
2. The initiator factor IF2 then binds to the Guanine-triphosphate (GTP) and to the
initiator fMet-tRNA to the P-site of the ribosomes.
3. A ribosomal protein splits the GTP that is bound to IF2 thus helping in driving the
assembly of the two ribosomal subunits. The IF3 and IF2 are released.

18 | P a g e
Translation Elongation
• The elongation of protein synthesis is aided by three protein factors i.e EF-Tu, EF-Ts,
and EF-G.
• The ribosomal function is known to shift one codon at a time, catalyzing the processes
that take place in its three sites.
• For every step, a charged tRNA enters the ribosomal complex and inserts the
polypeptides that become one amino acid longer, while an uncharged tRNA departs.
In prokaryotes, an amino acid is added at least every 0.05 seconds, which means that
about 200 polypeptide amino acids are translated in 10 seconds.

19 | P a g e
 • The bond created between each amino acid is derived from the Guanosine
Triphosphate (GTP), which is similar to Adenosine Triphosphate (ATP).
• The three sites (A, P, E) all participate in the translation process, and the ribosome itself
interacts with all the RNA types involved in translation.
• Therefore, three distinct steps are involved in translation, and these are;
1. The mediation of elongation Factor-Tu (EF-Tu) in the entry of amino-acyl-tRNAs to the
A site. This entails the binding of EF-Tu to GTP, which activates the EF-Tu-GTP complex
to bind to tRNA. The GTP then hydrolyses to GDP releasing an energy-giving phosphate
molecule, thus driving the binding of aminoacyl-tRNA to the A site. At this point the
EF-Tu is released, leaving the tRNA in the A-site.
2. Elongation factor EF-Ts then mediates the releasing of EF-Tu-GDP complex from the
ribosomes and the formation of the EF-Tu-GTP.
3. During this translocation process, the polypeptide chain on the peptidyl-tRNA is
transferred to the aminoacyl-tRNA on the A-site during a reaction that is catalyzed by
a peptidyl transferase. The ribosomes then move one codon further along the mRNA
in the 5′ to 3′ direction mediated by the elongation factor EF-G. This step draws its
energy from the splitting of GTP to GDP. Uncharged tRNA is released from the P-site,
transferring newly formed peptidyl-tRNA from the A-site to the P-site.
Translation Termination
• Termination of the translation process is triggered by an encounter of any of the three
stop codons (UAA, UAG, UGA). These triplets stop codons, however, are not recognized
by the tRNA but by protein factors known as the release factors, (RF1 and RF2) found
in the ribosomes.
• The RF1 recognizes the triplet UAA and UAG while RF2 recognizes UAA and UGA. A
third factor also assists in catalyzing the termination process and it’s known as Release
factor 3 (RF3).
• When the peptidyl-tRNA from the elongation step arrives at the P site, the release
factor of the stop codon binds to the A site. These releases the polypeptide from the
P site allowing the ribosomes to dissociate into two subunits by the energy derived
from GTP, leaving the mRNA.
• After many ribosomes have completed the translation process, the mRNA is degraded
allowing its nucleotides to be reused in other transcription reactions.

20 | P a g e
 Eukaryotes Protein Synthesis vs. Prokaryotes Protein Synthesis

S.N. Eukaryotes Prokaryotes

The mRNA for translation is monocistronic, The mRNA for translation is polycistronic, thus coding
1.
coding for a single gene of polypeptides for several genes of polypeptides

The three types of RNA polymerase are used for A single type of RNA polymerase is used to control the
2.
the synthesis of cellular RNA. synthesis of the types of RNA molecules

It involves both subunits of the ribosomes i. e

3. It involves 70S ribosomes
40S and 60S subunits.

Transcription and translation take place

4. Transcription and translation can overlap
separately hence they do not overlap.

The pre mRNA or an mRNA undergoes The mRNA doesn’t undergo any modification before
5.
modification before they are translated. translation.

They do have a special initiator complex of A special initiator tRNA Met-tRNAf or Met – tRNA is
6.
tRNA. used.

7. The starting amino acid is methionine. The starting amino acid is N-formyl methionine

They have a single initiation and termination

8. They have several initiation and termination sites.
site.

The ribosomal binding site (RBS) on mRNA is the Shine-

The Ribosomal Binding Site is Kozak sequence
9. Dalgarno sequence that lies -10 nucleotides ahead of
that is centered around the start codon
the initiation codon.

Several initiation factors are involved in initiating

10. the synthesise of the polypetide chain i.e eIF-2, It involves three initiation factors IF-1, IF-2, and IF-3.
(eIF-2, eIF-2al, eIF-a2, eIF-a

There are two chain elongation factors, EF-1 and There are three chain elongation factors, EF-Tu, EF-Ts,
11.
EF-2 and EP-G.

There is a single release factor eRF for

There are three release factors (RF-1 or RF-2 and RF-3)
12. recognition of three termination codons (UAA,
for recognition of termination codons.
UAG, and UGA).

21 | P a g e
 The genetic code may differ in mitochondria and The genetic code is the same in every prokaryotic
13.
chloroplast. organism.

Protein Synthesis Inhibitors

Antimicrobial agents are used as protein synthesis inhibitors which include:
1. Puromycin
• This is an antibiotic that is an analog of the terminal aminoacyl-adenosine part
of aminoacyl-tRNA. This antibiotic inhibits protein synthesis by releasing
prokaryotic polypeptides chains before they are completely synthesized.
2. Streptomycin
• This is a trisaccharide that has an effect on the binding activity of formyl
methionyl-tRNA to ribosomes. This prevents the correct initiation of protein
synthesis.
3. Aminoglycoside antibiotics such as neomycin, kanamycin, and gentamycin which
interfere with the decoding site in the 16s rRNA of the small subunit.
4. Chloramphenicol inhibits the activity of peptidyl transferase.
5. Erythromycin blocks translocation by binding to the 50S subunit
6. Cycloheximide is used to block peptidyl transferase in eukaryotic ribosomes and it is
used as a laboratory tool for blocking protein synthesis in eukaryotic cells.
7. Diphtheria toxin has an A fragment that catalyzes the transfer of a single side chain of
EF2 which blocks the translocation of the growing polypeptide chain.

Recombinant DNA Technology- Definition, Steps, Applications

• Recombinant DNA technology refers to the joining together of DNA molecules from
two different species that are inserted into a host organism to produce new genetic
combinations that are of value to science, medicine, agriculture, and industry.
• Recombinant DNA (rDNA), on the other hand is the general name for a piece of DNA
that has been created by the combination of at least two strands.
• They are DNA molecules formed by laboratory methods of genetic
recombination (such as molecular cloning) to bring together genetic material from
multiple sources, creating sequences that would not otherwise be found in
the genome.

22 | P a g e
Steps of Genetic Recombination Technology
1. Isolation of Genetic Material
• The first step in rDNA technology is to isolate the desired DNA in its pure form i.e. free
from other macromolecules.
• Since DNA exists within the cell membrane along with other macromolecules such as
RNA, polysaccharides, proteins, and lipids, it must be separated and purified which
involves enzymes such as lysozymes, cellulase, chitinase, ribonuclease, proteases etc.
• Other macromolecules are removable with other enzymes or treatments. Ultimately,
the addition of ethanol causes the DNA to precipitate out as fine threads. This is then
spooled out to give purified DNA.

23 | P a g e
 2. Restriction Enzyme Digestion
• Restriction enzymes act as molecular scissors that cut DNA at specific locations. These
reactions are called ‘restriction enzyme digestions’.
• They involve the incubation of the purified DNA with the selected restriction enzyme,
at conditions optimal for that specific enzyme.
• The technique ‘Agarose Gel Electrophoresis’ reveals the progress of the restriction
enzyme digestion.
• This technique involves running out the DNA on an agarose gel. On the application of
current, the negatively charged DNA travels to the positive electrode and is separated
out based on size. This allows separating and cutting out the digested DNA fragments.
• The vector DNA is also processed using the same procedure.
3. Amplification Using PCR
• Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA
sequence using the enzyme – DNA polymerase in vitro.
• It helps to amplify a single copy or a few copies of DNA into thousands to millions of
copies.
• PCR reactions are run on ‘thermal cyclers’ using the following components:
1. Template – DNA to be amplified
2. Primers – small, chemically synthesized oligonucleotides that are complementary to a
region of the DNA.
3. Enzyme – DNA polymerase
4. Nucleotides – needed to extend the primers by the enzyme.
• The cut fragments of DNA can be amplified using PCR and then ligated with the cut
vector.
4. Ligation of DNA Molecules
• The purified DNA and the vector of interest are cut with the same restriction enzyme.
• This gives us the cut fragment of DNA and the cut vector, that is now open.
• The process of joining these two pieces together using the enzyme ‘DNA ligase’ is
‘ligation’.
• The resulting DNA molecule is a hybrid of two DNA molecules – the interest molecule
and the vector. In the terminology of genetics this intermixing of different DNA strands
is called recombination.
• Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and
the technology is referred to as the recombinant DNA technology.

24 | P a g e
 5. Insertion of Recombinant DNA Into Host
• In this step, the recombinant DNA is introduced into a recipient host cell mostly, a
bacterial cell. This process is ‘Transformation’.
• Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to make
them ‘competent’ to accept new DNA. The processes used may be thermal shock,
Ca++ ion treatment, electroporation etc.
6. Isolation of Recombinant Cells
• The transformation process generates a mixed population of transformed and non-
trans- formed host cells.
• The selection process involves filtering the transformed host cells only.
• For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid
vector is employed.
• For examples, PBR322 plasmid vector contains different marker gene (Ampicillin
resistant gene and Tetracycline resistant gene. When pst1 RE is used it knock out
Ampicillin resistant gene from the plasmid, so that the recombinant cell become
sensitive to Ampicillin.
Application of Recombinant DNA technology

• Recombinant DNA is widely used in biotechnology, medicine and research.

• The most common application of recombinant DNA is in basic research, in which the
technology is important to most current work in the biological and biomedical
sciences.
• Recombinant DNA is used to identify, map and sequence genes, and to determine their
function.
• Recombinant proteins are widely used as reagents in laboratory experiments and to
generate antibody probes for examining protein synthesis within cells and organisms.
• Many additional practical applications of recombinant DNA are found in industry, food
production, human and veterinary medicine, agriculture, and bioengineering.

25 | P a g e