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CHEM 160

• Describe the following processes: replication,
transcription and translation, in terms of the
• Cellular localization
• Enzymes and other factors involved
• Sequence of reactions
• Physiological significance
Central Dogma of Molecular
Replication Biology
DNA Transcription

Transcription RNA


The Synthesis of DNA
• Process by which DNA makes identical copies of
• Occurs during the S-phase of the cell cycle in
preparation for cell division
• Characteristics:
1. Semiconservative
2. Bidirectional
3. Semi-discontinuous
The Cell Cycle

Campbell, 2009
Steps in DNA Replication
1. Unwinding of the two DNA strands
2. Formation of the replication fork
3. Synthesis of the RNA primer
4. Synthesis of New DNA strand
5. Excision of the RNA primer and replacement
with DNA
1. Separation of DNA Strands
• Begins at the origin of replication, a sequence
rich in A-T pairs
• Prokaryotes – single origin
• Eukaryotes – multiple origins
1. Separation of DNA Strands
2. Formation of Replication Fork
• DnaA Protein
• opens duplex at specific sites in origin
• SSBP (Single strand DNA-binding proteins)
• Binds to ssDNA and keeps them separated
• DnaB Protein (DNA helicase)
• Unwinds the DNA double helix
• DNA Topoisomerase (DNA gyrase)
• Relieves torsional strain brought about by
DNA unwiding
2. Formation of Replication Fork

3. Synthesis of RNA Primers
• RNA Primers
• Provides the free 3’―OH group to where
deoxyribonucleotides will be added
• Needed because the major DNA replicating
enzyme can only extend a pre-existing DNA
strand and cannot initiate a nucleotide
• RNA Primase
• Catalyzes the synthesis of the RNA primers
4. Chain Elongation
• DNA Polymerase III
• Catalyzes chain elongation
• Requires dATP, dGTP, dCTP, dTTP
• Also proofreads the new strand for
4. Chain Elongation
4. Chain Elongation
5. Excision of Primers and
Sealing of Nicks
• DNA Polymerase I
• Excises RNA primers and fills in gaps with
deoxyribonucleotides until only one
phosphodiester bond remains to be formed
• DNA ligase
• Catalyzes formation of phosphodiester bond
to seal nicks formed during elongation
Characteristics of Replication
• Semi-conservative
• Bidirectional
• Semidiscontinuous
1. Semi-conservative
• Daughter DNA
molecules contain
one strand from the
parent and one
newly synthesized
2. Bidirectional
• Occurs in two opposite directions about the
origin of replication
3. Semidiscontinuous
• Synthesis of new DNA strand always occurs
from the 5’ to 3’ direction with respect to the
DNA strand being synthesized
• The free 3’―OH group serves as the point of
attachment of incoming nucleotides during
3. Semidiscontinuous
3. Semidiscontinuous
• Leading strand
• continuous elongation
• Lagging strand
• elongation is discontinuous
• short chains are first made (Okazaki
fragments) which are eventually linked
together to form a long chain
3. Semidiscontinuous
The Synthesis of RNA
Flow of Genetic Information
• Genetic information stored in DNA
• Useful when expressed in the production of
RNA and functional proteins
• Gene expression
• RNA is synthesized from DNA
• RNA encodes directions/instructions for
protein synthesis
• Part of the genome (entire DNA) that encodes
for a particular functional protein
• Basic components of a (prokaryotic) gene:
• Promoter sequence
• RNA-coding sequence (first base = +1)
• Terminator sequence
• Copying of the genetic code
• Synthesis of mRNA using DNA as template
• Major enzyme involved is RNA polymerase
Features of Transcription
1. Specific base pairing
• Cytosine pairs with Guanine
• Adenine pairs with Uracil
2. Only one strand of the DNA is transcribed
• Template/anti-sense strand which is on the
3’ to 5’ direction
3. mRNA is synthesized in the 5’ to 3’ direction
Features of Transcription
4. mRNA is a copy of the sense strand, only that
T is replaced with U

Sense strand: 5’― A T G A C G A G T A A ―3’

Anti-sense strand: 3’― T A C T G C T C A T T ―5’

mRNA: 5’― A U G A C G A G U A A ―3’

RNA Polymerase
• Major enzyme involved in transcription
• DNA-directed RNA polymerase
• Uses DNA as template
• A multienzyme complex with five polypeptide
chains and a loosely held sigma factor
• Sigma (σ) factor is essential for accurate
binding of the RNA polymerase to the DNA
and recognition of the promoter sequences
Initiation of Transcription
• In the E. coli system, promoter regions
• “TATAA” box (−10 region, Pribnow box)
• “TTGACA” sequence (−35 region)

5’--------- TTGACA ---16-18 bases--- TATAA ---- ------3’
3’--------- AACTGT ---16-18 bases--- ATATT ---- ------5’
antisense −35 −10 +1
Initiation of Transcription
• RNA polymerase binds non-specifically to DNA
then migrates to the promoter sites
• Initial binding of the RNA polymerase with the
promoter site is called “closed promoter
• RNA polymerase then unwinds several base
pairs (open promoter complex) and initially
adds several nucleoside triphosphates (NTPs)
Initiation of Transcription
Initiation of Transcription
• After 8-9 nucleotides have been polymerized,
the sigma factor dissociates from the core
Elongation of mRNA
• RNA polymerase then moves along the
template DNA to continue elongation of the
growing mRNA chain
Elongation of mRNA
• RNA polymerase then moves along the
template DNA to continue elongation of the
growing mRNA chain
Termination of Transcription
Rho (ρ)-independent
• RNA polymerase
reaches a termination
signal which codes for
the RNA sequence that
forms a hairpin
• Hairpin causes
dissociation of the
mRNA, DNA and the
Termination of Transcription
Rho (ρ)-dependent
• ATP-dependent rho-
protein binds to the
nascent mRNA and pulls
it away from the RNA
polymerase and DNA
Nascent mRNA
• Has to go out of the
nucleus for translation
(for eukaryotes)
• Nascent mRNA has to
undergo post-
modifications to
protect it from being
degraded as it goes out
of the nucleus
Post-Transcriptional Processes
1. Splicing /removal of non-coding sequences
Post-Transcriptional Processes
• Alternative splicing results to multiple protein
Post-Transcriptional Processes
2. 5’ Capping and 3’ Poly-A Tailing

Capping with 7-
methyl guanosine

• Protects mRNA from the action of nucleases

when it goes out of the nucleus
Post-Transcriptional Processes
The Synthesis of
• Process wherein polypeptide chains are
• Specific ribonucleotide sequences in mRNA
form a message that determines the order
in which different amino acid residues are
to be joined
• Message carried by mRNA through codons is
translated into a specific amino acid
sequence characterizing a specific protein.
• A sequence of three ribonucleotides specific
for a given amino acid
The Genetic Code
Features of the Genetic Code
1. Non-overlapping
• Read in successive groups of three
2. Degenerate
• An amino acid can be coded for by more
than one codon (except Met and Trp)
3. Presence of start and stop codons
• Start: AUG (met)
• Stop: UAG, UGA, UAA
Requirements for Translation
1. Template - mRNA
2. Ribosome – site of translation
3. Amino acids
4. tRNA – carrier of the activated amino acid
5. Amino acid-tRNA synthetase
6. Initiation, elongation and termination factors
• Site of translation
• A site
• Aminoacyl tRNA
binding site
• P site
• Peptidyl tRNA
binding site
• E site
• Exit site
• Adaptor that links the codons and amino acids
• Each amino acid
has a specific
tRNA used for
• Enzyme
activation is also
specific for a
given amino acid
Steps in Translation
1. Activation of amino acids
2. Initiation
• Formation of initiation complex
3. Elongation of the polypeptide chain
• Binding, peptide bond formation, and
4. Termination
1. Activation of Amino Acids

Alberts et al 2008
1. Activation of Amino Acids
Amino acid + ATP → amino acyl-AMP + PPi
Amino acyl-AMP + tRNA → amino acyl-tRNA + AMP

• Catalyzed by: amino acyl-tRNA synthetase

• Eg. for the activation of alanine
• Enzyme: alanine-tRNA synthetase
• Activated form: alanine-tRNAAla
2. Initiation Complex Formation
1. Binding of mRNA and small ribosomal unit (30S)
• Shine-Dalgarno sequence complementary to
a portion of the 16S rRNA
• Facilitated by initiation factor 3 (IF3)

2. Binding of the first charged tRNA

• fmet-tRNAfmet
• Initiation factor 2 (IF2) hydrolyzes GTP to
provide energy for the association of the
mRNA, 30S ribosome and fmet-tRNAfmet
2. Initiation Complex Formation
3. Binding of the large ribosomal unit (50S)
• fmet-tRNAfmet on the P-site
• IF2 and IF3 dissociate from the initiation
2. Elongation of Polypeptide
1. Binding of the next charged amino acid
• A-site as entry point
• Facilitated by EF-Tu and requires hydrolysis
of GTP
2. Transfer of the polypeptide
• Polypeptide from the tRNA in the P-site
moves to the amino acid attached to the
tRNA in the A-site
2. Elongation of Polypeptide
3. Formation of peptide bond
• Catalyzed by peptidyl transferase
4. Translocation
• Promoted by another elongation factor EF-G
and requires GTP hydrolysis
• Places empty tRNA in the E-site
• tRNA with growing polypeptide chain now on
the P-site
• Next codon in the mRNA is on the A-site
3. Termination of Translation
1. Recognition of stop codons in the A-site

2. Binding of release factor (RF) in the A-site

• Cleaves the polypeptide chain from the tRNA

3. Dissociation of the translation machinery

CHEM 160 lecture slide of Profs. BP Serrano, KMP Caldo and AC Reyes

Nelson and Cox. Lehninger’s Principles of Biochemistry, 4th ed.

Stryer, Berg and Tymoczko. Biochemistry 4th ed.