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Agustin Krisna Wardani
Transcription in Prokaryotes
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Gene Express ion
1. Transcriptioncopies the information of a DNA
sequence (the gene) into corresponding
information in an RNA sequence.
2. Translationconverts this RNA sequence intothe amino acid sequence of a polypeptide.
The expression of a gene to form a polypeptide
occurs in two major steps:
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The central dogma of molecular biology
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The synthesis of RNA molecules using
DNA strands as the templates so that the
genetic information can be transferred
from DNA to RNA.
Transcription
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DNA vs RNA
RNA much shorterthan DNA
-DNA contains the code for making lots of different
proteins.
-Messenger RNA contains the information to make justone protein
DNA has two strands arranged in a double helix. RNA
consists of a single strand.
DNA (deoxyribonucleic acid) has a backbone of alternating
deoxyriboseand phosphate groups. In RNA (ribonucleicacid), the sugar ribosereplaces deoxyribose.
RNA uses the base uracil (U) rather than thymine (T):
A---U, G---C
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RNA (ribonucleic acid) has the sugar ribose
replaces deoxyribose.
RNA (RiboNucleic Acid)
DNA (DeoxyriboNucleic Acid)
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RNA uses the base uracil (U) rather than thymine (T):
The only difference between the two
molecules is the presence or absence of
the CH3group.
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Similarity between
replication and transcription
Both processes use DNA as the
template.
Phosphodiester bondsare formed in
both cases.
Both synthesis directionsare from5to 3.
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replication transcription
template double strands single strand
primer yes no
Enzyme DNA polymerase RNA polymerase
product dsDNA ssRNA
base pairA-T, G-C
The Amazing Gene Code (TAGC).A-U, T-A, G-C
Differences between
replication and transcription
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Transcriptional Control
DNA
RNA
protein
Environmental change
Turn gene(s) on/off
Proteins to deal withnew environment
Very important to:
1. express genes when needed
2. repress genes when not needed
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The wholegenomeof DNA needs to be
replicated, but only small portion of genome
is transcribedin response to the
development requirement, physiological
need and environmental changes.
DNA regions that can be transcribed into
RNA are called structural genes.
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Transcriptional Control
DNA
RNA
protein
TranscriptionInitiation
Elongation
Termination
ProcessingCapping
Splicing
Polyadenylation
Turnover
Translation
Protein processing
Many places for control
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Prokaryotic Transcription
Operons
Groups of related genes transcribed
by the same promoter
Polycistronic RNA
(one mRNA code several genes)
Multiple genes transcribed
as ONE TRANSCRIPT
No nucleus, so transcription and
translation can occur simultaneously
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Each transcriptable region is called
operon.
One operon includes several structural
genesand upstream regulatorysequences(or regulatory regions).
The promoteris the DNA sequence that
RNA-pol can bind. It is the key pointfor the transcription control.
Recognition of Origins
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Nisin Operon
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Template and Enzymes
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Template
The template strandis the strandfrom which the RNA is actuallytranscribed. It is also termed as
antisensestrand.
The coding strandis the strandwhose base sequence specifies the
amino acid sequence of the encodedprotein. Therefore, it is also called assensestrand.
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G C A G T A C A T G T C5' 3'
3' C G T C A T G T A C A G 5' templatestrand
codingstrand
transcription
RNAG C A G U A C A U G U C5' 3'
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RNA Polymerase
The enzyme responsible for the RNA
synthesis is DNA-dependent RNA
polymerase.
The prokaryotic RNA polymeraseis a
multiple-subunit protein of ~480kD.
Eukaryotic systemshave three kinds of
RNA polymerases I, II, III), each of which
is a multiple-subunit protein and
responsible for transcription of different
RNAs.
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Types of RNA
Messenger RNA (mRNA) genes that encodeproteins
Ribosomal RNA (rRNA) form the core ofribosomes
Transfer RNA (tRNA) adaptors that link
amino acids to mRNA during translation
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Transcriptional Control
TranscriptionInitiation
Elongation
Termination
ProcessingCapping
Splicing
Polyadenylation
Turnover
Translation
Protein processing
Control of initiation
usually most
important.
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Three phases: initiation, elongation,
and termination.
The prokaryotic RNA-polcan bind to
the DNA template directlyin the
transcription process.
The eukaryotic RNA-polrequires co-factorsto bind to the DNA template
together in the transcription process.
General concepts
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Initiation phase: RNA-pol recognizesthe promoter and starts thetranscription.
Elongation phase: the RNA strand iscontinuously growing.
Termination phase: the RNA-pol stopssynthesis and the nascent RNA isseparated from the DNA template.
Transcription of Prokaryotes
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a. Initiation
RNA-pol recognizesthe TTGACAregion, and slidesto the TATAATregion, then opensthe DNA duplex.
The unwound region is about 171 bp.
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Initiation
RNA polymerase
Promoter DNA
RNAP binding sites
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RNA Polymerase in prokaryote
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Initiation
RNA polymerase
4 core subunits
Sigma factor ()
determines promoter
specificity
Core + = holoenzyme
Binds promoter sequence
Catalyzes open complex and
transcription of DNA to RNA
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RNA Polymerase II in eukaryote
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Promoter
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The first thing that the enzyme has to do is to find the
start of the gene on the coding strand of the DNA.
These base sequences are known as promoter
sequences.
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5'
3'
3'
5'
regulatorysequences
structural gene
promotorRNA-pol
Promoter
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5'
3'
3'
5'-50 -40 -30 -20 -10 1 10
start -10region
T A T A A TA T A T T A
(Pribnow box)
-35
region
T T G A C AA A C T G T
Prokaryotic promoter
Consensus sequence
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Consensus Sequence
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The -35 region of TTGACAsequence
is the recognition siteand the
binding site of RNA-pol.
The -10 region of TATAATis the
region at which a stablecomplex of
DNA and RNA-pol is formed.
RNAP binds specific promoter
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RNAP binds specific promoter
sequences
Sigma factors recognize consensus
-10 and -35 sequences
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RNA polymerase promoters
TTGACA TATAAT
Deviation from consensus -10 , -35 sequence leads to
weaker gene expression
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Bacterial sigma factors
Sigma factors are transcription factors
Different sigma factors bind RNAP and recognize
specific -10 ,-35 sequences
Helps melt DNA to expose transcriptional start site Promote broad changes in gene expression
E. coli7 sigma factors
B. subtilis18 sigma factors
Generally, bacteria that live in more variedenvironments have more sigma factors
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Sigma factors
E. colican choose between 7 sigma factors and about 350
transcription factors to fine tune its transcriptional output
An Rev Micro Vol. 57: 441-466T. M. Gruber
Sigma subunit Type of gene controlled # of genes controlled
RpoD Growth/housekeeping ~1000
RpoN N2; stress response ~15
RpoS Stationary phase, virulence ~100
RpoH Heat shock ~40
RpoF Flagella-chemotaxis ~40
RpoE ? ~5
FecI Ferric citrate transport ~5
Extreme heat shock, unfolded proteins
70
54
S
S
F
32
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elongation
RNA-pol, DNA form a complex called
the transcription bubble.
The 3segmentof the nascent RNA
hybridizes with the DNA template, and
its 5endextends out thetranscription bubble as the synthesis
is processing.
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Once the enzyme has attached to the DNA, itunwinds the double helix over a short length, and
splits the two strands apart. This gives a "bubble" in
which the coding strand and template strand are
separated over the length of about 10 bases.
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Transcription bubble
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the enzyme moves along the DNA, zipping it up
again behind it. Essentially it moves the bubble
along the chain, adding new nucleotides all thetime. The growing RNA tail becomes detached from
the template strand as the enzyme moves along.
RNA s nthesis
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RNA synthesis
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Transcriptional Control
TranscriptionInitiation
Elongation
Termination
ProcessingCapping
Splicing
Polyadenylation
Turnover
Translation
Protein processing
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How does the enzyme know where to stop after it
reaches the end of the gene?
there will be a term inat ion sequenceof bases.
Once the enzyme gets to those, it stops adding new
nucleotides to the chain and detaches the RNA
molecule completely from the template chain.
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Transcriptional Termination
Bacteria need to end transcription at the
end of the gene
2 principle mechanisms of termination in
bacteria:
Rho-independent (more common)
Rho-dependent
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Rho-independent termination
Termination sequence has 2 features:
Series of U residues
GC-rich self-complimenting region
GC-rich sequences bind forming stem-loop
Stem-loop causes RNAP to pause
U residues unstable, permit release of RNA chain
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Rho-dependent termination
Rho is protein (50kDa) that terminatetranscription
Rho attach rut (rho utilization site),Binds the 70-80 base segment of RNA
Rho moves along RNA and catch up toRNAP, then unwinds DNA/RNA hybrid
Rho has ATPase activity which can
induce release of the polymerase fromDNA.
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RNA hairpins followed by a run of U residues in rho independent
terminator, but not in rho dependent terminator of transcription
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Quorum Sensing
Bacteria produce and secrete chemical signal
molecules (autoinducers)
Concentration of molecules increases with
increasing bacterial density When critical threshold concentration of
molecule is reached, bacteria alter gene
expression
Way for communities of bacteria to talk to
each other
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