DNA Replication
DNA replication represents the process in which a DNA double helix molecule forms two
new DNA molecules with the same sequence of bases as in the original DNA.
Replication of DNA is semiconservative: During replication, the two strands of the DNA
double helix molecule are separated. Each separated DNA strand is used as a template for the
synthesis of a new strand, resulting two new DNA molecules, each of them containing one strand
from the original parent DNA molecule and one newly synthesized strand.
The point where DNA replication starts is called origin of replication:
● in prokaryotic cells there is a single origin of replication;
● in eukaryotic cells there are multiple origins of replication.
The region where the supercoiled DNA molecule unwinds and opens to allow replication
is called the replication fork (it is a dynamic region: it moves along the DNA molecule as the
process of synthesis occurs).
Replication occurs in the same time on both DNA strands and only in the 5'-3' direction.
Replication fork in DNA
In the leading strand DNA synthesis is continuous, in the 5'-3' direction, the process
being catalyzed by the enzyme DNA polymerase III.
In the lagging strand DNA synthesis is discontinuous, in fragments (only in the 5'-3'
direction), called Okazaki fragments (the process being catalyzed by the same enzyme DNA
polymerase III), which are joined in the end of replication by the enzyme DNA ligase.
Stages of prokaryotic DNA replication
DNA replication occurs in four stages: prepriming, priming, elongation and
termination.
(initiation)
In the prepriming stage DNA is prepared for replication. In this step are involved
enzymes and proteins that recognize the origin of replication and/or the replication fork and
which are responsible for unwinding and opening the double helix DNA molecule and for
maintaining the separation of the parental strands (they form the prepriming complex).
● DnaA protein is the key component in the initiation of DNA replication: it recognizes a
specific sequence in origin of replication and opens the double helix in origin.
● Helicase (DNA helicase) or DnaB protein binds to single-stranded DNA near the
replication fork and unwinds the DNA molecule; then moves in the neighbouring double-stranded
region, unwinding the double helix. (Helicase requires energy provided by ATP)
● DnaC protein acts along with DnaB protein.
● SSB (single stranded binding) proteins bind only to single stranded DNA and are
responsible to keep the two strands of DNA separated, preventing reformation of the double
helix.
● DNA topoisomerases (I and II) are responsible for removing supercoils in the double
helix, relaxing in this way the supercoiled structure of DNA.
In the priming stage acts an enzyme called primase (it is a specific RNA polymerase) or
DnaG protein which synthesizes a short fragment of RNA called RNA primer (the RNA primer is
complementary and hydrogen-bonded to the DNA template).
In the elongation stage starts the synthesis of new DNA. In this stage acts another
enzyme called DNA polymerase III. This enzyme catalyzes DNA synthesis in the 5'-3' direction;
first it adds a deoxyribonucleotide to the RNA primer, followed by addition of other
deoxyribonucleotides to form a new DNA.
In the leading strand DNA synthesis is continuous, while in the lagging strand DNA
synthesis is discontinuous, in fragments called Okazaki fragments (the small DNA molecules
attached to their own RNA primer are called Okazaki fragments).
DNA polymerase III needs three components in this process of elongation: a DNA
template, all the four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, dTTP) and
magnesium ions (Mg+2).
DNA polymerase III also has a 3'-5' exonuclease activity (in addition to its 5'-3'
polymerase activity), which checks and removes incorrect added bases before DNA synthesis
continues.
Elongation stage in DNA replication
In the termination stage is involved DNA polymerase I, an enzyme which has two
important roles: to remove the RNA primer and to fill the resulting gaps with the complementary
deoxyribonucleotides.
DNA polymerase I also exhibits a 3'-5' exonuclease activity (it can eliminate any
incorrectly added base and substitute it with the correct base).
This is the end of the termination stage of DNA synthesis on the leading strand. On the
lagging strand, in the end of the termination stage, DNA ligase links the Okazaki fragments.
1.
2.
3.
The enzyme primase synthesizes RNA primers for the Okazaki fragments.
The RNA primers are extended by adding a DNA fragment in the presence of DNA
polymerase III.
DNA synthesis continues until the fragment extends as far as the primer of the
previously added Okazaki fragment.
Origin of replication of E. coli (ori C)
(Ori C has 245 base pairs which are highly conserved in bacterial replication origins; there are two series of sequences
important for initiation of replication: three 13 base pair sequences and four 9 base pair sequences)
1. The key component in the initiation of
DNA replication is the DnaA protein. About
20 molecules of DnaA protein bind at the four
9 base pair sequences and the DNA will be
wrapped around these molecules.
2. The three 13 base pair sequences (rich in
A=T pairs) are then denatured; DnaA protein
(in association with histone – like protein)
unwinds and opens ori C.
3. DnaB protein binds to each strand, with the
aid of DnaC protein and further unwinds the
DNA molecule.
DNA Transcription
Transcription represents the process of RNA synthesis directed by a DNA template; all
forms of RNA (mRNA, tRNA and rRNA) are produced through transcription.
In this process, the genetic information present in the double-stranded DNA molecule is
copied to a single-stranded RNA molecule. This means that only one strand of the double-stranded
DNA is transcribed into RNA. The DNA strand that is transcribed (called the template, sense or
copy strand) is the 3'-5' strand. The strand that is not transcribed (called the nontemplate or
antisense strand) is the 5'-3' strand.
Prokaryotic DNA transcription is catalyzed by RNA polymerase, a large protein with
five subunits.
Stages of transcription in prokaryotes
Transcription occurs in three stages (steps): initiation, elongation and termination.
In the initiation stage the RNA polymerase holoenzyme binds to the double-stranded
DNA at the promoter region (sequence) through the σ (sigma) subunit, which recognizes the
promoter region on the DNA molecule; after transcription begins, the σ subunit is released.
● In prokaryotes, the promoter region includes:
1) The Pribnow box or TATA box:
- It is rich in thymine and adenine;
- It contains six nucleotides: TATAAT;
- It is located at a distance of about 10 base pairs before the transcription
start site.
2) The –35 sequence:
- It is another sequence of six nucleotides: TTGACA;
- It is located at a distance of about 35 base pairs before the transcription
start site.
● In eukaryotes, the promoter region includes:
1) The Hogness box or TATA box:
- It is rich in thymine and adenine;
- It contains six nucleotides: ATATAA;
- It is located at a distance of about 25 base pairs before the transcription
start site.
2) The CAAT box:
- It is a sequence with eight nucleotides: GGCCAATC;
- It is located at a distance of about 75 base pairs before the transcription
start site.
Elongation stage: Once the promoter region has been recognized by the holoenzyme
(through the σ subunit), the σ subunit is released and RNA polymerase (through the core enzyme,
which has a polymerase activity) begins to synthesize an RNA chain. RNA polymerase uses
ribonucleoside triphosphates (ATP, GTP, CTP and UTP) in the process of elongation.
The DNA double helix is transiently unwound and opened as the RNA polymerase core
enzyme proceeds along the DNA. These local unwindings and openings of the DNA molecule are
necessary because DNA is a double helix structure, but in the process of transcription only one
strand of the DNA (the template strand) is used for synthesis of the RNA chain.
This continuous addition of ribonucleoside triphosphates by RNA polymerase using the
DNA template is called elongation. Elongation continues until a termination region is reached.
In the termination stage the RNA strand is released from the RNA polymerase and the
RNA polymerase core enzyme is also released from the DNA. Termination occurs when a
termination signal is reached; there are two types of termination signals:
1) Termination may occur when the RNA polymerase core enzyme encounters a DNA
region (termination region) which contains a palindromic sequence (palindrome). A palindrome
is a region of the DNA molecule in which each of the two strands has the same sequence of bases
when they are read in the same direction.
Example of a palindromic sequence
In the DNA of E. coli (Escherichia coli), the termination region is rich in A=T base pairs
with a palindromic sequence before the A=T rich region. Transcription of this termination region
signals RNA polymerase to stop transcription. This is ρ (rho)-independent termination, which
requires that the newly synthesised RNA to have two important characteristics:
A. The newly synthesized RNA must be able to form a stable “hairpin” structure (rich in
G≡C base pairs) before the end of the RNA chain. This hairpin structure may disrupt the RNA –
DNA interactions and/or the interactions between the RNA strand and the RNA polymerase,
facilitating the dissociation of the RNA chain.
B. Following the hairpin structure, the newly formed RNA molecule must contain a short
sequence of uracil (U). The bonding of uracil (U) to the corresponding adenine (A) on the DNA
template is weak (two hydrogen bonds), facilitating the separation of the RNA transcript from the
DNA template.
2) Termination may occur in the presence of a termination factor called ρ (rho) protein
or ρ (rho) factor, which signals the end of transcription. This ρ (rho) factor is attached to the
DNA at the termination region and stops the RNA polymerase, which cannot move further. The
enzyme (RNA polymerase) dissociates from DNA and RNA is released. This is ρ (rho)dependent termination (it is important when stable hairpin structures are not formed).
Structure of the prokaryotic promoter region
Structure of the eukaryotic promoter region
RNA Translation (Protein Synthesis)
replication
transcription
DNA
translation
RNA
PROTEIN
The “central dogma” of molecular biology
Translation is a cytoplasmic process in which the sequence of bases in the mRNA directs
the synthesis of a protein. Protein synthesis (translation) occurs in ribosomes.
mRNA is the template that directs the sequence of amino acids incorporated into a
protein. The codons of mRNA (sequences of three bases in mRNA) are determined by the
sequence of bases on the DNA template.
tRNA transfers each amino acid to the correct site in the growing protein. Each tRNA
carries a specific amino acid. The tRNA molecules contain a sequence of three bases called
anticodon, which recognizes a specific codon on the mRNA.
rRNA is a part of the ribosome structure, where protein synthesis takes place. The
ribosomes are complex structures, each of them having two subunits, a small and a large one: the
30S and 50S ribosomal subunits of prokaryotes form the 70S ribosome; in eukaryotes, the 40S
and 60S ribosomal subunits form together the 80S ribosome.
Stages of translation in prokaryotes
The synthesis of proteins in prokaryotes can be divided into four stages: activation,
initiation, elongation and termination.
1)
In the activation stage an amino acid binds to a specific tRNA. This stage has two steps:
a) Activation of the amino acid: In this step the carboxyl group of an amino acid binds to
the phosphate group of AMP (derived from the hydrolysis of ATP) to form an “activated”
amino acid (AA – AMP).
ATP
← H2O
→ PPi
AA + AMP → AA – AMP
b) Transfer of the activated amino acid to its specific tRNA: In this step the amino acid
residue of AA – AMP complex is transferred to a tRNA specific for that amino acid,
resulting an AA – tRNA complex with liberation of AMP.
AA – AMP + tRNA → AA – tRNA + AMP
Both reactions are catalyzed by the same enzyme: aminoacyl – tRNA synthetase.
2)
In the initiation stage are involved the following components:
the two ribosomal subunits of prokaryotes (30S and 50S)
the mRNA, required as a template for protein synthesis
the AA – tRNA complex formed in the activation stage – it is the tRNA which carries
the initiating (N-terminal) amino acid
GTP, which provides energy for the process
initiation factors (IF1, 2, and 3) – are proteins that facilitate the assembly of this
initiation complex
AUG, the initiation codon on the mRNA, codes for the amino acid methionine (Met). In
the prokaryotic cells, N-formyl methionine (fMet), a formylated methionine, is the first amino acid
synthesized in the proteins of all prokaryotes (in eukaryotes, the initiator tRNA carries a
methionine that is not formylated); it is brought to the AUG codon by a special tRNA molecule,
which carries N-formyl methionine to the ribosome.
Initially, the fMet-tRNA complex combines with other components to form the 30S
initiation complex (which contains mRNA, fMet-tRNA, the 30S ribosomal subunit and the
initiation factors); then, the 50S ribosomal subunit is added to the 30S initiation complex to form
the functional 70S initiation complex.
Initiation stage in translation
The initiation AUG codon is guided to its correct position on the 30S ribosomal subunit,
where it is required for initiation of translation, by a purine-rich sequence in the mRNA known as
the Shine-Dalgarno sequence. This sequence, located at about 10 bases before the initiation AUG
codon on the mRNA near its 5'-end, pairs with a complementary pyrimidine-rich sequence near
the 3'-end of the 16S rRNA of the 30S ribosomal subunit. The initiation AUG codon, where the
fMet-tRNA complex will be bound, is distinguished from other similar codons by its proximity to
the Shine-Dalgarno sequence.
Shine-Dalgarno sequence on the mRNA that serves as a signal for initiation of translation in prokaryotes
3)
In the elongation stage the amino acids are added one by one to form a protein. This stage
has three steps:
a) Aminoacyl-tRNA binding. At the start of this step the fMet-tRNA complex is bound to the
P (peptidyl) site of the ribosome. The other site of the ribosome – the A (aminoacyl) site – is
empty. In this step, the second amino acid (AA2), carried to the ribosome by a specific
tRNA (as an AA2 – tRNA complex) and specified by the next codon of the mRNA, binds to
the A site.
b) Peptide bond formation. In this step the amino acid fMet dissociates from the fMettRNA complex and forms a peptide bond with the second amino acid at the A site of the
ribosome. The result is that an uncharged tRNA occupies the P site and a dipeptidyltRNA occupies the A site.
c) Translocation. In this step three events occur:
- the uncharged tRNA leaves the P site
- the dipeptidyl-tRNA moves from the A site to the P site
- mRNA moves a distance of three nucleotides
After translocation the A site is empty, ready to receive the next AA-tRNA complex to
start another round of elongation. The sequence of aminoacyl-tRNA binding, peptide bond
formation and translocation repeats until all of the amino acids, specified by the mRNA codons
and carried by tRNA, are joined by peptide bonds to form a polypeptide chain. Elongation
protein factors (EF – Tu, EF – Ts, EF – G) and GTP (as a source of energy) are required in this
process of elongation.
4)
The termination stage marks the end of protein synthesis. The signal for the termination
stage is given by the appearance of a termination codon (or stop codon) – UAA, UAG or UGA
– in the A site of the ribosome. Protein release factors (RF1, RF2) recognize and bind to one of
these termination codons and cause hydrolysis of the newly synthesized protein from its tRNA.
The nascent protein, the uncharged tRNA and mRNA are released and leave the ribosome.
Finally, the ribosome dissociates into its 30S and 50S subunits and all components are then
available for another cycle of translation.
Note: RF1 recognizes the termination codons UAA or UAG. RF2 recognizes the termination
codons UAA or UGA. There is a third protein release factor – RF3 – but the specific function of
RF3 is not exactly known, although it is thought to release the ribosomal subunit and stimulates
the activity of RF1 and RF2.
Genetic code
The genetic code is the sequence of codons in mRNA that determines the sequence of
amino acids in a protein. A codon is a set of three bases in mRNA that codes for a specific amino
acid.
Characteristics of the genetic code:
1. The genetic code is a triplet code because three bases (one codon) specifies one amino
acid.
2. The genetic code is universal (with a few minor exceptions): the genetic code has been
highly preserved during evolution (the codons are the same for the same amino acid in all
species).
3. The genetic code is degenerate because the four RNA bases (A, G, C, U) can combine
into 64 different codons. Because there are only 20 amino acids to encode and 64 possible base
combinations, this means that an amino acid can be coded by more than one codon.
4. The genetic code is commaless: every base is part of a codon and there are no “free”
bases which do not participate in any codon.
5. The genetic code is nonoverlapping because no bases are shared between consecutive
codons; each base is in a sequence of bases is part of only one codon.
6. Only 61 of the 64 codons specify amino acids. The three remaining codons (UAA,
UAG and UGA) are termination codons (or stop codons) and they do not code for any amino
acid.
7. AUG is the codon for methionine and in all the polypeptide chains synthesized through
the process of translation, AUG acts as an initiation codon.
8. Each codon has only one meaning.
The genetic code (composed of 64 codons)