Features of DNA replication
DNA Replication
1. Semi conservative
ENZYME: DNA -directed DNA polymerase 2. Ordered and sequential
3. Uses activated substrates
4. Semi discontinuous
5. Accurate
DNA RNA Protein
Three Steps for Replication
1. Initiation
2. Elongation
3. Termination
INITIATION: Prokaryotic DNA Replication
A circular bacterial chromosome
has a single origin of replication.
oriC: the origin of replication of
E. Coli
DNA Synthesis occurs inside the nucleus 1. Recognition of the origin
of replication by specific
proteins
2. Formation of replication
fork
Proteins involved in the
formation of replication fork
Dna A for identification of the origin of
replication
DNA Helicase unwinds the DNA
Single-Stranded DNA binding protein prevents
the reannealing of DNA
DNA polymerase in prokaryotes Discontinuous synthesis of DNA in lagging strand
Mechanism of DNA synthesis
Complications in DNA replication
1. Both strands of the DNA double helix must be
copied. DNA polymerase has only 5’ 3’
polymerase activity.
2. DNA polymerase cannot initiate DNA synthesis
on a molecule that is entirely single-stranded.
ELONGATION: Prokaryotic DNA Replication
Formation of Primosome
Formation of Replisome
1. Excision of RNA primers by DNA polymerase I Transcription and Translation
2. Filling up of the gaps by DNA polymerase I
3. Nick ligation Gene Expression in an outline:
1. Accessing the genome
2. Assembly of the transcription initiation complex
3. Synthesis of RNA
4. Processing of mRNA
5. RNA degradation
6. Assembly of the translation initiation complex
7. Protein synthesis
8. Protein folding and protein processing
9. Protein degradation
DNA polymerase has a proofreading ability
DNA polymerase III
3’ 5’ exonuclease
activity
DNA polymerase I
3’ 5’ exonuclease
activity
5’ 3’ exonuclease
activity
What is TRANSCRIPTION? Mammalian cells possess three distinct nuclear DNA-
dependent RNA polymerases.
ENZYME: DNA-dependent RNA polymerase
Compartment Genes Transcribed
RNA Nucleolus 28S, 5.8S and 18S
polymerase I ribosomal RNA
(rRNA)
genes
RNA Nucleoplasm Protein-coding
polymerase II genes; most small
nuclear
RNA (snRNA)
genes
RNA Nucleoplasm Genes for transfer
polymerase III RNAs (tRNA), 5S
rRNA,
U6-snRNA, small
nucleolar (sno)
RNAs,
Transcription vs Replication small cytoplasmic
(sc) RNA
Transcription Replication
Enzyme DNA-dependent DNA-dependent
RNA polymerase DNA polymerase Three Phases of Transcription
Substrate NTPs dNTPs
Template 3’ 5’ 5’ 3’ Phase 1: INITIATION phase in Prokaryotes
3’ 5’
Primer No primer Requires Primer
Two complementary DNA strands have different roles
in transcription.
1. Template (antisense) strand
The strand that serves as the template for
RNA synthesis. 1. Pribnow Box /-10 region
2. Coding (nontemplate/sense) strand 2. -35 region
It is identical to the RNA transcribed 3. UP element
from the gene, with U in the RNA in Recognition of
place of T in the DNA. promoter region by
Bacterial DNA-dependent RNA polymerase is a multi- σ factor (closed
subunit enzyme. promoter complex)
RNA polymerase
It is made up of five core subunits (α2ββ’ω, bind to DNA
about 400kDa) and a sixth subunit, σ factor. Unwinding of the
o The β subunit is thought to be the DNA (open
catalytic subunit. promoter complex)
o The σ factor directs the core complex to
specific binding sites on the DNA.
Phase 2: ELONGATION phase in Prokaryotes
Addition of purine ribonucleotide triphosphate
as the first base of the RNA transcript.
RNA polymerase initiates mRNA synthesis
Dissociation of σ factor, elongation proceeds
Prokaryotic mRNAs are synthesized on the bacterial
nucleoid in direct contact with the cytosol and are
immediately available for translation.
Phase 3: TERMINATION phase in Prokaryotes
ρ-Dependent Mechanism (Extrinsic Termination)
ρ factor recognizes the termination region
It weakens the interaction between the
template and the transcript.
Eukaryotic mRNAs are processed while they are being
synthesized.
Addition of 5’ capping
Splicing of introns
Polyadenylation at 3’ tail
Capping of Eukaryotic mRNA
ρ-Independent Mechanism (Intrinsic Termination) Addition of GTP by guanylyl transferase
Methylation of G by guanine methyltransferase
Termination sites contains palindromes in the
template strand
Newly synthesized RNA form a ‘hairpin’ loop
What is TRANSLATION?
The synthesis of
polypeptides using
the genetic code
from RNA.
Splicing of introns
The GENETIC CODE
Introns are made up of GU-AG motifs. specifies how an
o 5′ splice site 5′-AG↓GUAAGU-3′ mRNA sequence is
o 3′ splice site 5′-PyPyPyPyPyPyNCAG↓-3′ translated to a
polypeptide.
Steps in Splicing pathway
1. Cleavage of the 5′ splice site
2. Cleavage of the 3′ splice site and joining of the
exons.
Five Major Features of the Genetic Code
1. Triplet
2. Nonoverlapping
3. Commaless
4. Degenerate
5. is not Universal!
Spliceosome-mediated intron splicing
Spliceosome is made up of snRNAs associated
with snRNPs.
Requirements for Translation
1. RNAs (mRNA, tRNA, rRNA)
2. Ribosome
3. Protein Factors (IFs, EFs, RFs)
4. Activated Substances
The role of tRNA in Translation
It serves as an adaptor molecule, that provides physical
and informational link between mRNA and the
polypeptide being synthesized.
Structure of tRNA:
Acceptor arm
D arm
Anticodon loop
V loop
TψC arm
Aminoacylation: Charging of tRNA
Codon-Anticodon Interaction
“Wobble” base pairing is a nonstandard base pairing
between the base at 5’ end of anticodon and the base
at 3’ end of codon.
Phases of Translation Shine-Dalgarno sequence
1. Activation phase
2. Initiation
3. Elongation
4. Termination
Elongation
Events in the elongation and termination phase are
similar for bacteria and eukaryotes.
Initiation in Prokaryotes Three Steps:
Formation of Initiation Complex 1. aa-tRNA binding
mRNA 2. Peptide bond formation
30S & 50 S ribosomal unit 3. Translocation
fmet-tRNAfmet
GTP, Mg+2
IF-1, IF-2, IF-3
Termination
Release factors recognize stop codons
RF-1 – recognizes 5′-UAA-3′ and 5′-UAG-3′,
RF-2 - recognizes 5′-UAA-3′ and 5′-UGA-3′
RF-3 – stimulates the dissociation of RF1 and
RF2
Posttranslational Processing and Modifications
A polypeptide chain (a polymer of amino acids)
that must be folded into its proper three-
dimensional form and in some cases, disulfide
bonds must be formed
Chemical modification (eg. proline to
hydorxyproline)
Covalent modification
Glycosylation – addition of carbohydrate moiety
