Replication

1. Basic Overview of DNA Structure

 DNA Composition: DNA (Deoxyribonucleic Acid) is a molecule
composed of two long strands forming a double helix. It
contains the genetic blueprint for building and maintaining an
organism.
 Nucleotides: Each strand is made of nucleotides, which are
the building blocks of DNA. A nucleotide consists of three
parts:
 A phosphate group
 A deoxyribose sugar
 A nitrogenous base (Adenine [A], Thymine [T], Cytosine
[C], Guanine [G])
2. The Double Helix Structure
 Strand Orientation: The two strands of DNA run in opposite
directions, known as antiparallel. One strand runs in the 5' to 3'
direction, while the other runs 3' to 5'.
 Base Pairing: The nitrogenous bases pair specifically:
Adenine pairs with Thymine (A-T), and Cytosine pairs with
Guanine (C-G). These pairs are held together by hydrogen
bonds—A-T by two hydrogen bonds and C-G by three.
3. Detailed DNA Replication Process
 Semiconservative Replication: DNA replication is called
semiconservative because each new DNA molecule consists of
one original strand and one newly synthesized strand.
 Steps in DNA Replication:
1. Initiation:
 Origin of Replication: Replication begins at
specific locations on the DNA called origins of
replication.
 Unwinding the Double Helix: The enzyme
helicase unwinds the double helix by breaking the
hydrogen bonds between base pairs, forming a
replication fork.
 Single-Strand Binding Proteins (SSBs): These
proteins bind to the separated strands to prevent
them from rejoining.
2. Elongation:
 Primase and Primer: Primase synthesizes a short
RNA primer on the template strand, which provides
a starting point for DNA synthesis.
 DNA Polymerase: This enzyme adds nucleotides
to the 3' end of the RNA primer, extending the DNA
strand in the 5' to 3' direction.
  Leading and Lagging Strands:
 Leading Strand: Synthesized continuously
toward the replication fork.
 Lagging Strand: Synthesized discontinuously
away from the replication fork in short
fragments called Okazaki fragments.
 DNA Ligase: After RNA primers are replaced with
DNA, DNA ligase seals the gaps between the
Okazaki fragments to create a continuous strand.
3. Termination:
 Replication Ends: The process continues until the
entire molecule is replicated. Enzymes like
topoisomerase prevent the DNA from becoming
too coiled during replication.
 Proofreading and Repair: DNA polymerase also
has a proofreading ability to ensure the accuracy of
DNA replication by correcting any mismatched
bases.
4. Significance of the Double Helix in
Replication
 The double helix structure ensures that each strand contains
all the information necessary to replicate the complementary
strand. This redundancy allows cells to reproduce their genetic
material accurately during cell division, ensuring that each
daughter cell receives an exact copy of the DNA.
This process is essential for growth, repair, and reproduction in
living organisms.

Transcription
 Transcription: The process by which the genetic information
in DNA is copied into RNA. It is the first step in gene
expression.
 Prokaryotes: Organisms like bacteria that lack a true nucleus
and membrane-bound organelles.
 Eukaryotes: Organisms like plants, animals, and fungi that
have a true nucleus and membrane-bound organelles.
Key Steps in Transcription
1. Initiation:
 Prokaryotes: Transcription begins when RNA
polymerase binds to the promoter region of the DNA. The
sigma factor helps the RNA polymerase recognize the
promoter.
 Eukaryotes: Multiple transcription factors are required
for RNA polymerase II to recognize and bind to the
 promoter (especially the TATA box). The DNA unwinds,
forming a transcription bubble.
2. Elongation:
 Prokaryotes: RNA polymerase synthesizes the RNA
strand by adding nucleotides complementary to the DNA
template strand. It moves along the DNA, unwinding it
and re-winding it as it goes.
 Eukaryotes: RNA polymerase II synthesizes the pre-
mRNA strand, and as it does, the nascent RNA strand
detaches from the DNA. Eukaryotic cells also exhibit RNA
processing during elongation, such as capping at the 5'
end.
3. Termination:
 Prokaryotes: Transcription ends when RNA polymerase
encounters a terminator sequence. This can be a simple
sequence that causes the RNA to fold into a hairpin
structure, leading to the detachment of RNA polymerase.
 Eukaryotes: Termination is more complex and involves
a sequence known as the polyadenylation signal. After
this signal, the RNA transcript is cleaved, and a poly-A tail
is added to the 3' end of the RNA.
Differences Between Prokaryotic and Eukaryotic
Transcription
1. Location:
 Prokaryotes: Transcription occurs in the cytoplasm
since they lack a nucleus.
 Eukaryotes: Transcription occurs inside the nucleus, and
the mRNA must be transported to the cytoplasm for
translation.
2. RNA Polymerase:
 Prokaryotes: A single type of RNA polymerase
transcribes all types of RNA (mRNA, tRNA, rRNA).
 Eukaryotes: There are three main types of RNA
polymerase: RNA polymerase I (rRNA), RNA polymerase II
(mRNA), and RNA polymerase III (tRNA and other small
RNAs).
3. RNA Processing:
 Prokaryotes: mRNA is often translated as soon as it is
transcribed, with little to no modification.
 Eukaryotes: The pre-mRNA undergoes extensive
processing, including 5' capping, splicing (removal of
introns), and 3' polyadenylation before it becomes
mature mRNA and is exported from the nucleus.
4. Genes Structure:
  Prokaryotes: Genes are often organized in operons (a
group of genes regulated together), leading to
polycistronic mRNA, which codes for multiple proteins.
 Eukaryotes: Genes are typically monocistronic (one
gene per mRNA), and each gene has its own promoter
and terminator.
Summary
 Prokaryotic transcription is simpler and quicker, reflecting
the simpler cellular structure and need for rapid response to
environmental changes.
 Eukaryotic transcription is more complex due to the
presence of introns and exons, the need for RNA processing,
and the spatial separation of transcription and translation.

Translation
Translation is the process by which the genetic code carried by mRNA (messenger RNA) is
used to synthesize proteins in cells. This process occurs in the cytoplasm of eukaryotic cells
and involves several key components, including mRNA, tRNA (transfer RNA), ribosomes,
and various enzymes.

1. Overview of Translation

Translation is the final step of gene expression, where the information encoded in mRNA is
translated into a specific sequence of amino acids to form a protein. The mRNA, which was
transcribed from DNA in the nucleus, carries the code in the form of codons, which are
sequences of three nucleotides. Each codon specifies a particular amino acid.

2. Charging of tRNA

 tRNA: Transfer RNA is a small RNA molecule that carries amino acids to the
ribosome during translation. Each tRNA has an anticodon region that is
complementary to a specific mRNA codon.
 Aminoacyl-tRNA Synthetase: This enzyme is responsible for "charging" the tRNA.
Charging refers to the process where an amino acid is attached to its corresponding
tRNA.
o Step 1: The enzyme binds to both the amino acid and the corresponding
tRNA.
o Step 2: The amino acid is activated by ATP (adenosine triphosphate), forming
an aminoacyl-AMP (aminoacyl adenylate) intermediate.
o Step 3: The activated amino acid is then transferred to the 3' end of the tRNA,
forming an aminoacyl-tRNA (charged tRNA).
o Step 4: The enzyme releases the charged tRNA, now ready to participate in
translation.

3. Structure of the Ribosome

  Ribosomes: Ribosomes are the molecular machines that facilitate the translation of
mRNA into protein. They are made up of two subunits: the large subunit and the
small subunit.
o Small Subunit (40S in eukaryotes): This subunit is responsible for binding
the mRNA and ensuring correct base-pairing between the mRNA codon and
the tRNA anticodon.
o Large Subunit (60S in eukaryotes): This subunit contains the active sites for
peptide bond formation and has the A (aminoacyl), P (peptidyl), and E (exit)
sites.
o APE Sites: These sites are located within the large subunit and are crucial for
the translation process.
 A Site (Aminoacyl-tRNA site): The A site is where the charged tRNA
enters the ribosome and base-pairs with the mRNA codon.
 P Site (Peptidyl-tRNA site): The P site holds the tRNA carrying the
growing polypeptide chain.
 E Site (Exit site): The E site is where the uncharged tRNA, having
donated its amino acid to the growing chain, exits the ribosome.

4. Initiation of Translation

 Start Codon: The translation process begins at the start codon, which is typically
AUG, coding for the amino acid methionine in eukaryotes.
o Step 1: The small ribosomal subunit binds to the mRNA at the 5' end and
scans along the mRNA until it encounters the AUG start codon.
o Step 2: The charged initiator tRNA (carrying methionine) base-pairs with the
start codon at the P site.
o Step 3: The large ribosomal subunit then assembles with the small subunit,
forming a functional ribosome ready for elongation.

5. Elongation

 Step 1: Codon Recognition: The next charged tRNA, corresponding to the mRNA
codon at the A site, enters the ribosome. Its anticodon base-pairs with the mRNA
codon.
 Step 2: Peptide Bond Formation: The ribosome catalyzes the formation of a peptide
bond between the amino acid on the tRNA in the P site and the amino acid on the
tRNA in the A site. This bond formation transfers the growing polypeptide chain to
the tRNA at the A site.
 Step 3: Translocation: The ribosome moves (or translocates) along the mRNA by
one codon, shifting the tRNA with the growing peptide chain from the A site to the P
site. The uncharged tRNA in the P site moves to the E site and is then released from
the ribosome.
 Step 4: The process repeats, with a new charged tRNA entering the A site, until a stop
codon is reached.

6. Termination
  Stop Codon: Translation ends when a stop codon (UAA, UAG, or UGA) is
encountered in the mRNA. Stop codons do not code for any amino acid and do not
have corresponding tRNAs.
o Release Factors: Proteins known as release factors bind to the stop codon in
the A site, triggering the release of the polypeptide chain from the tRNA in the
P site.
o Disassembly: The ribosome subunits, mRNA, and tRNA disassemble, and the
newly synthesized polypeptide is released into the cytoplasm where it can fold
into its functional form.

7. Post-Translation Modifications

 Once the polypeptide chain is synthesized, it often undergoes further modifications,

such as folding, cleavage, and the addition of chemical groups (like phosphorylation
or glycosylation) to become fully functional.

This entire process ensures that the genetic information stored in DNA is accurately
expressed as proteins, which perform various functions in the cell, from catalyzing reactions
to providing structural support.