tRNA Structure

The tRNAs are generally 76-90 nucleotides long. Each amino acid has specific tRNAs. Stop
codons are not recognised by any tRNAs. The secondary structure of tRNA looks like a clover
leaf and tertiary structure is like an inverted ‘L’ shape. The folded structure is formed due to
hydrogen bonding between complementary bases.

Clover leaf structure of tRNA

The secondary folded structure of tRNA has three hairpin loops, which give it an appearance
of three-leafed clover. The main constituents of tRNA are:

Acceptor arm

It is formed by the base pairing of 7-9 nucleotides of 5’ terminal and 3’ terminal. The 5’
terminal has a phosphate group and the 3’ ends with a specific sequence of CCA or CCA tail.
The amino acid attaches to the 3’ hydroxyl group of the acceptor arm.

The aminoacylation of tRNA or charging of tRNA is the first step of the translation process.
The enzyme aminoacyl tRNA synthetases catalyse the reaction.
DHU Loop

D arm has a stem of 3-4 base pairs and it ends in a loop called D loop as it generally contains
dihydrouridine, a modified nucleotide.

Anticodon Loop

It has a 5 base pair long stem. It has an anticodon loop, which contains the complementary
codon (3 nucleotides sequence) present on mRNA for the amino acid it carries. These unpaired
bases of anticodon loop pair with the mRNA codon. Each codon is identified by a specific
tRNA.

TΨC Loop

The T arm consists of a stem of 4-5 bp and a loop containing pseudouridine, modified uridine.

Variable Loop

It is present between the TΨC loop and the anticodon loop. Its size varies from 3-21 bases. It
helps in the recognition of the tRNA molecule.

tRNA Function

 tRNA plays an important role in protein synthesis.

 It acts as an adapter molecule for linking amino acids to its specific codon present in
mRNA.
 Aminoacylation of tRNA is the first step in protein synthesis.
 tRNA is specific to each amino acid and carries them during the translation process in
the ribosomal subunits.
 The tRNA transfers the amino acid to the growing polypeptide chain in the ribosomes,
which has three binding sites for tRNA, namely A, P and E, which correspond to
aminoacyl, peptidyl and exit, respectively.
 This decoding of codons of mRNA by specific tRNAs continues until the entire
sequence for a polypeptide chain is translated.
 DNA Replication
In the process of DNA replication, the DNA makes multiple copies of itself. It is a biological
polymerisation, which proceeds in the sequence of initiation, elongation, and termination. It is
an enzyme-catalysed reaction. DNA Polymerase is the main enzyme in the replication process.
Role of Enzymes in DNA Replication
DNA replication is a highly enzyme-dependent process. There are many enzymes involved in
DNA replication, which includes the enzymes, DNA-dependent DNA polymerase, helicase,
ligase, etc. Among them, DNA-dependent DNA polymerase is the main enzyme.

DNA Replication Steps

Following are the important steps involved in DNA replication:

Initiation

DNA replication demands a high degree of accuracy because even a minute mistake would
result in mutations. Thus, replication cannot initiate randomly at any point in DNA.
For the replication to begin there is a particular region called the origin of replication. This is
the point where the replication originates. Replication begins with the spotting of this origin
followed by the unwinding of the two DNA strands.

Unzipping of DNA strands in their entire length is not feasible due to high energy input. Hence,
first, a replication fork is created catalysed by the helicase enzyme, which unzips the DNA
strand.

Elongation

As the strands are separated, the polymerase enzymes start synthesising the complementary
sequence in each of the strands. The parental strands will act as a template for newly
synthesising daughter strands.

It is to be noted that elongation is unidirectional i.e. DNA is always polymerised only in the 5′
to 3′ direction. Therefore, in one strand (the template 3‘→5‘) it is continuous, hence called
continuous replication while on the other strand (the template 5‘→3‘) it is discontinuous
replication. They occur as fragments called Okazaki fragments. The enzyme called DNA ligase
joins them later.

Termination

Termination of replication occurs in different ways in different organisms. In E.coli like

organisms, chromosomes are circular. And this happens when the two replication forks
between the two terminals meet each other.

DNA-dependent DNA polymerase

It helps in the polymerisation, catalyses and regularises the whole process of DNA replication
with the support of other enzymes. Deoxyribonucleoside triphosphates are the substrate as well
as the energy provider for the replication process. DNA polymerase is of three types:

DNA Polymerase I

It is a DNA repair enzyme. It is involved in three activities:

 5′-3′ polymerase activity

 5′-3′ exonuclease activity
 3′-5′ exonuclease activity

DNA Polymerase II

It is responsible for primer extension and proofreading.

DNA Polymerase III

It is responsible for in vivo DNA replication.

DNA Replication Process in Prokaryotes

The DNA replication in prokaryotes takes place in the following place:

1. The two strands of DNA unwind at the origin of replication.

2. Helicase opens the DNA and replication forks are formed.
3. The DNA is coated by the single-strand binding proteins around the replication fork
to prevent rewinding of DNA.
4. Topoisomerase prevents the supercoiling of DNA.
5. RNA primers are synthesised by primase. These primers are complementary to the
DNA strand.
6. DNA polymerase III starts adding nucleotides at the end of the primers.
7. The leading and lagging strands continue to elongate.
8. The primers are removed and the gaps are filled with DNA Polymerase I and sealed
by ligase.

DNA Replication in Eukaryotes

The DNA replication in eukaryotes is similar to the DNA replication in prokaryotes. However,
the initiation process is more complex in eukaryotes than prokaryotes. In eukaryotes, there are
multiple origins of replication present. A pre-replication complex is made with other initiator
proteins. The process is entirely the same but the enzymes used are different. E.g. in eukaryotes,
the polymerisation process is carried out by the enzyme Pol δ, whereas in prokaryotes it is done
by DNA Pol III.
 Transcription
The process of synthesis of RNA by copying the template strand of DNA is called transcription.
During replication entire genome is copied but in transcription only the selected portion of
genome is copied. The enzyme involved in transcription is RNA polymerase. Unlike DNA
polymerase it can initiate transcription by itself, it does not require primase. More exactly it is
a DNA dependent RNA polymerase.
Steps of transcription
Transcription is an enzymatic process. The mechanism of transcription completes in three
major steps
1. Initiation:
 closed complex formation
 Open complex fromation
 Tertiary complex formation
2. Elongation
3. Termination
 Rho- dependent
 Rho-independent

1. Initiation:
 The transcription is initiated by RNA polymerase holoenzyme from a specific
point called promotor sequence.
 Bacterial RNA polymerase is the principle enzyme involved in transcription.
  Single RNA polymerase is found in a bacteria which is called core polymerase
and it consists of α, β, β’ and ω sub units.
 The core enzyme bind to specific sequence on template DNA strand called
promotor. The binding of core polymerase to promotor is facilitates and
specified by sigma (σ) factor. (σ70 in case of E. coli).
 The core polymerase along with σ-factor is called Holo-enzyme ie. RNA
polymerase holoenzyme.
 In case of e. coli, promotor consists of two conserved sequences 5’-TTGACA-
3’ at -35 element and 5’-TATAAT-3’ at -10 element. These sequence are
upstream to the site from which transcription begins. Binding of holoenzyme to
two conserve sequence of promotor form close complex.
 In some bacteria, the altered promotor may exist which contain UP-element and
some may contain extended -10 element rather than -35 element.

2. Elongation:
 After synthesis of RNA more than 10 bp long, the σ-factor is ejected and the
enzyme move along 5’-3’ direction continuously synthesizing RNA.
 The synthesized RNA exit from RNA exit channel.
 The synthesized RNA is proof reads by Hydrolytic editing. For this the
polymerase back track by one or more nucleotide and cleave the RNA removing
the error and synthesize the correct one. The Gre factor enhance this proof reading
process.

3. Termination:
There are two mechanism of termination.
i.Rho independent:
 In this mechanism, transcription is terminated due to specific sequence in
terminator DNA.
 The terminator DNA contains invert repeat which cause complimentary pairing
as transcript RNA form hair pin structure.
 This invert repeat is followed by larger number of TTTTTTTT(~8 bp) on
template DNA. The uracil appear in RNA. The load of hair pin structure is not
tolerated by A=U base pair so the RNA get separated from RNA-DNA
heteroduplex.
ii.Rho dependent:
 In this mechanism, transcription is terminated by rho (ρ) protein.
 It is ring shaped single strand binding ATpase protein.
 The rho protein bind the single stranded RNA as it exit from polymerase
enzyme complex and hydrolyse the RNA from enzyme complex.
 The rho protein does not bind to those RNA whose protein is being
translated. Rather it bind to RNA after translation.
 In bacteria transcription and translation occur simultaneously so the rho
protein bind the RNA after translation has completed but transcription is
still ON.
Prokaryotic vs Eukaryotic Transcription

Prokaryotic Transcription Eukaryotic Transcription

Transcription and translation occur Transcription and translation don’t occur

simultaneously simultaneously.

Prokaryotic transcription occurs in the Eukaryotic transcription occurs in the

cytoplasm nucleus and translation occurs in the
cytoplasm.

RNAs are released and processed in RNAs are released and processed in the
the cytoplasm nucleus

RNA polymerases are a complex of RNA polymerases are a complex of 10 -15

five polypeptides. polypeptides.

Doesn’t require any proteins or other Requires proteins known as transcription

factors for the initiation of factors for the initiation.
transcription