Dr.

Nora Fahmy, PhD

Professor of Microbiology
Microbial

Genetics
 Review Questions
• Which one of the following components of DNA
replication involves multiple RNA primers?

A. Unwinding DNA double helix

B. Leading-strand DNA synthesis

C. Lagging-strand DNA synthesis

D. Repair of miss-incorporated DNA

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• ………….enzyme introduces supercoiling and packing

of DNA.

A. Topoisomerase type Ι

B. Topoisomerase type ΙΙ

C. Helicase

D. Ligase
 Central Dogma Theory
1. DNA contains the information
required for the synthesis of
another identical molecule of
DNA (Self-replication).
2. DNA contains all information
required for the synthesis of
RNA's (Transcription).
3. Some RNA's (mRNA) transmit
the information, which direct
protein synthesis (Translation).
 DNA
Replication
 DNA
Transcription
 Codons on mRNA
• The sequence of nucleotides on mRNA is read in
triplets (three letters) by the translation system
(ribosome an tRNA’s).

• A triple base sequence is called a codon.

• Each codon encodes or specifies a single amino

acid.
 How Many Codons??
• As mentioned before, mRNA composed from
four bases (A,U,G,C) which are arranged in
special sequence.
• The number of possible combinations of triplet
bases can be calculated as 4Ʌ3=64.
• A total of 64 different codons were found in most
examined living organisms.
• There are three codons which can not be encodes any
amino acid ……..Stop signals or stop codons (non-
sense codons).
• The appearance of any one of these three stop codons
during protein synthesis bring process to halt
(complete stop).
• These three codons are: UAA,UAG, and UGA.
• On other hand AUG is a start signal or start codon.
• AUG codon specifies the amino acid methionine in
eukaryotes and N-formyl derivative in bacteria.
• The remaining 60 codons specify only 19 amino
acids.
• Actually all amino acids (except methionine) have
several corresponding codons (leucine has
6 corresponding codons).
• This phenomenon is known as code degeneracy.
The Genetic Code
 Open Reading Frame
• Simply, Open Reading Frame (ORF) is a sequence on
DNA contains information of transcription of mRNA and
contains the sequential order.
• It requires a continuous sequence of DNA from a start
codon, through a subsequent region which usually has
a length that is a multiple of 3 nucleotides, to a stop
codon in the same reading frame
(Start codon + long base sequence + non-sense codon)
 Depending on the starting point,

In general, at the most, one reading frame in a given section of

a nucleic acid, is biologically relevant (open reading frame.
Some viral transcripts can be translated using multiple,
overlapping reading frames.
▪ "Open" means that the road is open to keep reading, and
the ribosome will be able to keep reading the RNA code and
add another amino acid one after another.
Post Transcriptional Modification

mRNA capping

Polyadenylation

Intron Splicing
• In case of eukaryotes, pre-mRNA molecule
undergoes three main modifications.
1. Five-prime cap (5′ cap)[mRNA capping]: methylated
guanosine is connected to mRNA via an unusual 5′ to 5′
triphosphate linkage.

2. Polyadenylation is the addition of a poly(A) tail to a

messenger RNA at 3' end. The poly(A) tail consists of
multiple adenosine monophosphates.
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• In eukaryotes, DNA transcription occurs in a cell's

nucleus. The RNA that is synthesized in this process is
then transferred to the cell's cytoplasm where it is
translated into a protein.
• And these modifications are required for such transport
and to avoid enzymatic degradation of transcripted RNA
in the cytoplasm.
• Because bacteria have no defined nucleus, thus RNA’s
are formed in the cytoplasm. So such transport
modifications are not required.
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3. Intron Splicing: The pre-mRNA transcripts often contain

introns, which are non-coding sequences that interrupt
the coding regions known as exons.

• Introns are removed and exons are joined together by the

spliceosome and specialized enzymes in the nucleus.

• This process occurs before translation process.

• On the other hand, bacterial DNA contains no such non-

coding introns. Therefore, such splicing enzymes are
absent in bacteria.
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(UTR)
• The key difference between coding sequence (CDS) and ORF is
that CDS is that actual nucleotide sequence of a gene which
translates into a protein while ORF is a stretch of DNA
sequence that begins with translation initiation site (start
codon) and ends with a translation termination site (stop
codon).
• A gene has a coding sequence (CDS). It consists of
total exons of the gene and a start codon and a stop codon.
It is the actual part of the gene that translates and produces
the protein.
• Open reading frame or ORF is a nucleotide sequence located
between a start codon and a stop codon. There is no stop
codon inside an ORF interrupting the genetic code which
translates into a protein.
• In prokaryotes, CDS and ORF of a gene are the same.
• All CDSs are ORFs. But not all ORFs are CDSs.

 tRNA: Transfer RNA
• Transfer RNA (abbreviated tRNA) is a small RNA
molecule that plays a key role in protein synthesis.

• Transfer RNA serves as a link (or adaptor) between

the messenger RNA (mRNA) molecule and the
growing chain of amino acids that make up a protein.

• It is a single stranded RNA made of 73-93 nucleotides.

• There is intra-strand base pairing (double strand) in

some regions giving tRNA cloverleaf structure.
• The tRNA molecule has a variable (in sequence) triplet
base region known as anticodon.
• Anticodons recognize the complementary triplet base
on mRNA.
• The function of tRNA is to guide the translation of the
code on a mRNA into protein.
• There are about 61 known tRNA types in bacteria.
• Each type of tRNA carries a specific amino acid
depending on its type of anticodon.
• Aminoacylation is the process of
adding an aminoacyl group to a
compound.
• It produces a tRNA molecule with
its CCA 3' end covalently linked
to an amino acid.
• Each aminoacylated tRNA
(charged ) with a specific amino
acid. This occurs by aminoacyl
tRNA synthetase.
Uncharged tRNA Charged tRNA

Charged tRNAs are tRNA molecules that have formed a bond with their
specific amino acid, while uncharged tRNAs do not have an amino acid
attached to them.
During the process of tRNA 'charging,' each tRNA molecule is linked to its
correct amino acid by a group of enzymes called aminoacyl tRNA synthetases.
 
 rRNA: Ribosomal RNA
• Ribosomal RNA (rRNA), molecule in cells that forms part
of the protein-synthesizing organelle known as
a ribosome and that is exported to the cytoplasm to help
translate the information in mRNA into protein.

• The ribosome is composed of rRNA and protein.

• It is made of two sub-units (large and small).

• These subunits generally are named according to their

rate of sedimentation, measured in Svedberg units [S].
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• Three sites in ribosome:

 A site: aminoacyl tRNA site.

 P site: peptidyl tRNA site.

 E site: exit site.

Translation
 Central Dogma Theory
1. DNA contains the information
required for the synthesis of
another identical molecule of
DNA (self-replication).
2. DNA contains all information
required for the synthesis of
RNA's (transcription).
3. Some RNA's (mRNA) transmit
the information, which direct
protein synthesis (translation).
Translation: Protein Synthesis
Initiation

Elongation

Termination
• Peptidyl transferase enzyme.
• A translocase enzyme.
 Initiation
• Protein synthesis begins in a 30s ribosomal subunit.
• mRNA binds to 30s ribosome by 6-8 bases [binding site;
sequence on the mRNA just upstream of the start
codon].
• The Shine–Dalgarno (SD) sequence is a ribosomal
binding site in bacterial and archaeal (but not
eukaryotic) messenger RNA, generally located around
8 bases upstream of the start codon AUG.
• The RNA sequence helps recruit the ribosome to
the messenger RNA (mRNA) to initiate protein
synthesis by aligning the ribosome with the start codon.
• The Shine–Dalgarno sequence was proposed
by Australian scientists John Shine and Lynn Dalgarno in
1973.

• Binding site is followed by the start codon (AUG).

• N-formyl-methionine-tRNA binds to AUG on mRNA.

• The complex formed of 30s subunit, mRNA, and formyl-

methionine-tRNA is called 30s initiation complex.
• GTP hydrolysis provides energy for binding of 50s subunit to
initiation complex to form 70s ribosome that embraced mRNA and
formylmethionine-tRNA.

• Note that formylmethionine-tRNA is now attached to AUG by

anticodon in 30s, but the rest of tRNA molecule and its amino acid
are attached to the P site on 50s subunit of the 70s ribosome.
The efficient interaction of mRNA with aminoacyl tRNA’s inside the
ribosome leads to efficient synthesis of the final protein.

• A special group of proteins participate in initiation and are thus

known as initiation factor.
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Large
ribosomal
subunit

P site
3 U
A C
5
5 A U G 3

Initiator tRNA
GTP GDP
E A
mRNA

5 3 5 3
Start codon

mRNA binding site Small Translation initiation complex

ribosomal
subunit
1 2
A small ribosomal subunit binds to a molecule of The arrival of a large ribosomal subunit completes
mRNA. In a prokaryotic cell, the mRNA binding site the initiation complex. Proteins called initiation
on this subunit recognizes a specific nucleotide factors (not shown) are required to bring all the
sequence on the mRNA just upstream of the start translation components together. GTP provides
codon. An initiator tRNA, with the anticodon UAC, the energy for the assembly. The initiator tRNA is
base-pairs with the start codon, AUG. This tRNA in the P site; the A site is available to the tRNA
carries the amino acid methionine (Met). bearing the next amino acid.
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Elongation
• The second aminoacyl tRNA binds strongly to the
A site in 50s subunit.
• Its anticodon react by hydrogen bond on with the
codon next to AUG on mRNA. Now two aminoacyl
tRNA are next to each other.
• Peptidyl transferase enzyme in 50s subunit catalyzes
the peptide bond formation between the two
adjacent amino acids.
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• N-formylmethionine-tRNA becomes uncharged because

N-formylmethionine has moved to form a dipeptide
with the amino acid of tRNA in A site.

• A translocase enzyme hydrolyze GTP to provide energy

required for moving the dipeptidyl-tRNA from the A site
to P site. This movement leads to the ejection of
uncharged tRNA from the complex.

• The A site is now vacant. A third aminoacyl tRNA

hydrogen bonds the third codon on mRNA.
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• The above steps are repeated that catalyzed by peptidyl

transferase and a tripeptide is formed.

• Elongation step is repeated in successive steps by the

addition of one amino acid at a time on the growing
peptide chain.

• Special proteins known as elongation factors participate

in the binding of aminoacyl –tRNA’s to the A site.
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Peptidyl transferase
A translocase enzyme enzyme
• Elongation factor thermal unstable Tu (EF-Tu) is a GTP-
binding protein that catalyzes the binding of aminoacyl-
tRNA to the A-site of the ribosome inside living cells.

• EF-G (elongation factor G, historically known

as translocase) is a prokaryotic elongation factor
involved in mRNA translation.

• As a GTPase, EF-G catalyzes the movement

(translocation) of transfer RNA (tRNA) and messenger
RNA (mRNA) through the ribosome.
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Termination
• The final step in translation is termination.

• When the ribosome reaches a STOP codon, there is

no corresponding transfer RNA.

• Instead, a small protein called a “release factor”

attaches to the stop codon.

• The release factor causes the whole complex to fall

apart: messenger RNA, the two ribosome subunits,
the new polypeptide.
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Polyribosomes
• The mRNA can be translated many times, to
produce many protein copies.

• A number of ribosomes can translate a single mRNA

molecule simultaneously forming a polyribosome.

• Polyribosomes enable a cell to make many copies of

a polypeptide very quickly.
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