CENTRAL DOGMA

Lecturer : Dra Lusia Hayati, MSc.

Department of Medical Biology
Faculty of Medicine,
Sriwijaya University
 Learning Objectives
•Be able to describe DNA replication, transcription
and translation
•Be able to define: promoter; initiation, elongation,
termination, rho factor; sigma factor, core and
holoenzyme RNA polymerases; -10 and -35
regions in transcription
•Be able to differ: transcription in eukaryotes from
that in prokaryotes
•Be able to define: codon, triplet,degenerate,
initiation and termination as they relate to the code
in translation
Transcription
Transcription

Splicing

Translation
Central dogma
 Transcription
(animation)

Transcription occurs in the nucleus

Factors involved in transcription:
 DNA template
 Transcription factors
 RNA polymerase
 ATP
 Nucleotides: ATP, UTP, GTP, CTP
RNA synthesis is in 5’ to 3’ direction
RNA strand is complementary to DNA template
Steps of Transcription

1. Initiation
2. Elongation
3. Termination
 Initiation

TFs

• Transcription factors (TFs) bind to promoter region (TATA box) of

target gene
• RNA polymerase binds to promoter
• TFs hydrolyze ATP to obtain energy for DNA unwinding
• RNA polymerase unwinds the DNA template
Promoters in Eukaryote
Promoters in Prokaryote (E. coli)
PROMOTER
 Properties of Promoters

• Promoters typically consist of 40 bp region

on the 5'-side of the transcription start site
• Two consensus sequence elements:
– The "-35 region", with consensus TTGACA
– The Pribnow box near -10, with consensus
TATAAT
RNA Polymerases in Eukaryote

• RNA polymerase I: rRNA synthesis

• RNA polymerase II: mRNA synthesis
• RNA polymerase III: tRNA synthesis

In prokaryote, there is only one RNA polymerase

RNA Polymerase in Bacteria
 Elongation

• Unwinding of double-stranded DNA in front of the enzyme

• Synthesis of RNA
• RNA proofreading
• Dissociation of RNA
• Re-annealing of DNA behind the enzyme
 Termination

• Dissociation of RNA strand from DNA template

• Dissociation of RNA polymerase from DNA template
RNA Splicing
 RNA Splicing
(animation)
• RNA modification which remove introns
and joined exons
• A convertion from "primary transcript"
to "mature mRNA"

• Intron: a DNA region within a gene that is not translated into protein

• Exon: a DNA region within a gene that is translated into protein

 The Eukaryotic Message

A. Finishing the ends

1. cap - GTP added backwards
2. polyA tail added enzymatically on
most messages.
Initiation of Transcription
1. RNA polymerase binds to DNA molecule at promoter
a. Pribnow (TATAAT) box at -10 (synthesis start signal)
b. upstream (TGTTGACA) at -35 (docking signal)

2. polymerase binds tightly at the -35

3. polymerase unwinds transcription bubble, region of about

17 base pairs, starting from the -10 base.
Elongation of
Transcription
1. Polymerase copies template (sense) strand only. The non-
template (antisense) strand is not copied, but it has the
same base sequence as the RNA (except for T's instead
of U's).

2. Synthesis proceeds in the same direction as DNA

synthesis - adding to 3' OH (5' to 3').

3. Polymerase uses nucleotide triphosphates, clipping off

terminal two phosphates as the phosphodiester bond is
formed.

4. Terminal two phosphates leave together as a

pyrophosphate, but are clipped apart by a separate
enzyme (pyrophosphorylase) afterward.
Termination of Transcription

1. signaled by GC rich sequence followed by 6 or

more A's.
2. signal translates in message into an inverted
repeat which produces a hairpin loop, followed
by a terminal run of U's copied from the A's.
3. A second type of signal does not used the above
but rather a sequence (the rut sequence) that
must be recognized by an additional protein
factor called rho
Termination (in prokaryote)
Rho-independent (intrinsic) terminator
 - 20 nucleotides inverted repeat sequence
 8 A:T bases

Rho-dependent terminator
 Rho factor bind to mRNA and dissociate from
RNA polymerase

The termination mechanism in eukaryote

is not well understood
Structure of a Termination site
Termination of Transcription
 Types of RNA
 Three types of RNA resulted from transcription:
– mRNA (produced by RNA polymerase II)
• Template for protein synthesis
– rRNA (produced by RNA polymerase I)
• Component of ribosomes
– tRNA (produced by RNA polymerase III)
• Essential factor for translation

mRNA
rRNA
tRNA
TRANSLATION
 Translation
(animation)

 Translation occurs in cytoplasm

 Factors involved in translation:
• mRNA
• Ribosome (large & small subunits)
• tRNA
• Release factor
 Translation steps:
• Initiation
• Elongation
• Termination
Mature mRNA

Ribosome
 The Nobel Prize in Chemistry 2009

Venkatraman Ramakrishnan Thomas A. Steitz Ada E. Yonath

Yale University Weizmann Institute of Science
MRC Laboratory of Molecular Biology
New Haven, CT, USA Rehovot, Israel
Cambridge, United Kingdom

Studies on structure
and function of
ribosomes
Do you know that some antibiotics
block the ribosome?
Release factor
Steps of Translation

1. Initiation
2. Elongation
3. Termination
 Initiation

 The initiator tRNA is formylated methionine: f-Met-tRNAiMet

 N-formyl methionine is first aa of all proteins
 Initiation of Translation
 1. Message binds to small (30s / 40s)
subunit of ribosome, using IF3
 2. met-tRNA or f-met tRNA binds to small
subunit of ribosome with help of IF2 and
GTP
 3. AUG (or alternative initiation codon)
comes up in reading frame. The met-tRNA
anticodon binds to message
 4. Large subunit binds to small, using GTP
for energy
 Elongation

Amino acids

Addition of amino acids based on codon in mRNA sequence

 Elongation
Steps to form each peptide bond
1. Correct next amino acyl tRNA binds, aided by EF-Tu
and GTP

2. Fidelity of match is checked (GTP hydrolyzed). The

energy from the GTP powers a jiggle in the A site

3. Peptide bond formed as previous amino acyl tRNA

detached from amino acid. Catalyzed by 28s or 23s
rRNA of the large (50s / 60s) subunit

4. Empty tRNA leaves

5. 3-base translocation (shift) brings next codon into

frame ( EF- G uses GTP)
 Termination

Binding of Releasing of Releasing of

release factor to tRNA from ribsosome from
stop codon mRNA strand mRNA strand
Translation Cycle
Gene Expression
 Codon Table
A set of nucleotide sequences (3 bases of DNA or RNA)
which encode one amino acid
• From RNA to polypeptide
• All 3 RNA types participate in the reaction:
• mRNA – template
• tRNA – adaptor
• rRNA - catalyst

• Genetic code
• 20 AA in animal cells
• 2 bases codon – 42 = 16 combinations
• 3 bases codon – 43= 64 combinations
• Each amino acid is specified by 3
nucleotides in mRNA
• 3 nucleotides form codon

• Degenerate
• Non-overlapping
• triplet
 Genetic code is read from fixed point
in a sequential manner

• Deletion of a nucleotide abolish function of

a specific gene; function may be restore by
insertion of a nucleotide in the vicinity of
primary mutation site
• Reading frame
• The genetic code has no internal
punctuation, to indicate the reading frame
• Sequence is read sequentially, triplet by
triplet
 Nature of the genetic code

 Code is highly degenerative (6 codons for Arg,

Leu and Ser)
 Synonymous codons – specify one AA
 Trp and Met – only one codon
 Synonymous codons differ in third nucleotide
(codons evolved to minimize the effect of
mutation)
 Stop codons: UAG, UAA, UGA; nonsense codon
 AUG and GUG – for Met and Val, also initiation
codon
Universality of the genetic code
 genetic code is almost universal
 majority of organism employs the same genetic
code
E. coli is able to correctly transcribed genes for
other organisms

Exceptions: certain mitochondria shows variant in

the genetic code

 AUG and AUA – Met and Start codon

protozoa: alternate genetic code

 Transfer RNA

1. Carries specific AA and

recognizes codon on
mRNA

2. tRNA structure:

5’ end is phosphorylated
Acceptor stem – AA is covalently
attached to 3’end;
3’end – CCA sequence, stem
contains non Watson-Crick
base pairs
D arm contains dihydrouridine
Anticodon arm
TC arm involves pseudouridine
Variable arm – 3 to 21 nucleotides modified
 Aminoacyl-tRNA synthetase
Catalyzes attachment of AA to 3’
end to tRNA forming aa-tRNA

a) correct AA must be selected

b) correct tRNA has to be

recognized
Esterification takes place at 3’-
OH or 2’-OH group

“activated amino acid” charged tRNA

Isoacepting tRNAs –
tRNAs carrying the same aa
 i) activation of AA residue

 ii) formation of aa-tRNA

Aminoacyl –AMP + tRNA + ATP  aa-tRNA + PPi + AMP