Transcription and Translation

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
What is TRANSCRIPTION?
ENZYME:
DNA-dependent
RNA polymerase
Transcription vs Replication
DNA-dependent DNA-dependent
RNA polymerase DNA polymerase
NTPs dNTPs
3’  5’ 5’  3’
3’  5’
No primer Requires for a
primer
Two complementary DNA strands have
different roles in transcription.
1. Template (antisense) strand

 The strand that serves as the template for RNA
synthesis.
2. Coding (nontemplate/sense) strand

 It is identical to the RNA transcribed from the gene,
with U in the RNA in place of T in DNA.
Bacterial DNA-dependent RNA polymerase
is a multi-subunit enzyme.
 It is made up of five core subunits (α2ββ’ω, about
400kDa) and a sixth subunit, σ factor.
 The β subunit is thought to be the catalytic subunit.
 The σ factor directs the core complex to specific binding
sites on the DNA.
Mammalian cells possess three distinct
nuclear DNA-dependent RNA polymerases.
Compartment Genes Transcribed
RNA polymerase I Nucleolus 28S, 5.8S and 18S ribosomal RNA (rRNA)
genes
RNA polymerase II Nucleoplasm Protein-coding genes; most small nuclear
RNA (snRNA) genes
RNA polymerase III Nucleoplasm Genes for transfer RNAs (tRNA), 5S rRNA,
U6-snRNA, small nucleolar (sno) RNAs,
small cytoplasmic (sc) RNA
RNA polymerase II
consists of 12 subunits.
(RPB1-RPB12)
Three Phases of Transcription
1. Initiation
2. Elongation
3. Termination
Phase 1: INITIATION phase in Prokaryotes
Promoter Regions in Prokaryotes

1. Pribnow Box /-10 region
2. -35 region
3. UP element
Phase 1: INITIATION phase in Prokaryotes
 Recognition of promoter
region by σ factor
(closed promoter complex)
 RNA polymerase bind to DNA
 Unwinding of the DNA
(open promoter complex)
Phase 2: ELONGATION phase in Prokaryotes
 Addition of purine
ribonucleotide triphosphate as
the first base of RNA transcript
 RNA polymerase initiates
mRNA synthesis
 Dissociation of σ factor,
elongation proceeds
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.
Phase 3: TERMINATION phase in Prokaryotes
ρ-Independent Mechanism (Intrinsic Termination)
 Termination sites contains palindromes in the
template strand
 Newly synthesized RNA form a ‘hairpin’ loop
Prokaryotic mRNAs
are synthesized on
the bacterial
nucleoid in direct
contact with the
cytosol and are
immediately
available for
translation.
Eukaryotic mRNAs are processed while
they are being synthesized.
 Addition of 5’ capping
 Splicing of introns
 Polyadenylation at 3’ tail
Capping of Eukaryotic mRNA
 Addition of GTP by guanylyl

transferase
 Methylation of G by guanine
methyltransferase
Splicing of introns
 Introns are made up of GU-AG motifs .
5′ splice site 5′-AG↓GUAAGU-3′
3′ splice site 5′-PyPyPyPyPyPyNCAG↓-3′
Steps in Splicing pathway

1. Cleavage of the 5′ splice
site
2. Cleavage of the 3′ splice
site and joining of the
exons
Spliceosome-mediated intron splicing
 Spliceosome is
made up of snRNAs
associated with
snRNPs.
What is TRANSLATION?
The synthesis of
polypeptides using
the genetic code
from RNA.
The GENETIC CODE specifies how an mRNA
sequence is translated to a polypeptide.
Five Major Features of the Genetic Code

1. Triplet
2. Nonoverlapping
3. Commaless
4. Degenerate
5. is not Universal!
The Genetic Code
Requirements for Translation
1. RNAs (mRNA, tRNA, rRNA)
2. Ribosome
3. Protein Factors (IFs, EFs, RFs)
4. Activated Substances
RNA
polypeptide
The role of tRNA in Translation
 It serves as 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 non-
standard base pairing
between the base at 5’ end of
anticodon and the base at 3’
end of codon.
The “wobble” base pairing

The Structure of Ribosome
Phases of Translation
1. Activation phase
2. Initiation
3. Elongation
4. Termination
Formation of Initiation Complex
 mRNA
 30S & 50 S ribosomal unit
 fmet-tRNAfmet
 GTP, Mg+2
 IF-1, IF-2, IF-3
Initiation in Prokaryotes
Shine-Dalgarno sequence
Initiation in Prokaryotes
Events in the elongation and termination phase are
similar for bacteria and eukaryotes.
Three Steps:
1. aa-tRNA binding
2. Peptide bond formation
3. Translocation
Elongation
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
Termination
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

Transcription and Translation

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Transcription and Translation

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Gene Expression in an outline

1. Accessing the genome

1. Template (antisense) strand

2. Coding (nontemplate/sense) strand

Promoter Regions in Prokaryotes

ρ-Dependent Mechanism (Extrinsic Termination)

 Addition of GTP by guanylyl

Steps in Splicing pathway

Five Major Features of the Genetic Code

The “wobble” base pairing

 A polypeptide chain (a polymer of amino acids) that

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