Transcription in Prokaryotes: Dindin H. Mursyidin Laboratory of Molecular Biology Lambung Mangkurat University

Transcription in Prokaryotes
Lecture 4
Dindin H. Mursyidin
Laboratory of Molecular Biology

Lambung Mangkurat University
Transcriptional Control
DNA Environmental change
Turn gene(s) on/off

RNA
protein
Proteins to deal with
new environment
Very important to:
1. express genes when needed
2. repress genes when not needed
3. Conserve energy resources; avoid expressing unnecessary/detrimental genes
Many places for control

Transcription
DNA Initiation
Elongation
Termination
Processing
RNA Capping
Splicing
Polyadenylation
Turnover
protein
Translation
Protein processing
Prokaryotic Transcription
 Operons
Groups of related genes transcribed
by the same promoter
 Polycistronic RNA
 Multiple genes transcribed

as ONE TRANSCRIPT
 No nucleus, so transcription and

translation can occur simultaneously
RNA Structure
 Contain ribose instead of deoxyribose

 Bases are A,G,C,U,
 Uracil pairs with adenine
 Small chemical difference from DNA,
but large structural differences
 Single stranded helix
 Ability to fold into 3D shapes - can be
functional
RNA Structures Vary
 RNA more like proteins than DNA:

structured domains connected by more flexible
domains, leading to different functions
 e.g. ribozymes – catalytic RNA
RNA synthesis •
 RNAP binds, melts DNA
 Nucleosides added 5’  3’
Types of RNA
 Messenger RNA (mRNA) – genes that encode
proteins
 Ribosomal RNA (rRNA) – form the core of

ribosomes
 Transfer RNA (tRNA) – adaptors that link

amino acids to mRNA during translation
 Small regulatory RNA – also called non-

coding RNA
Transcription
Initiation
Elongation
Termination
Control of initiation
usually most Processing
Capping
important. Splicing
Polyadenylation
Turnover
Translation
Protein processing
Initiation
 RNA polymerase α α β β’σ

 Transcription factors
 Promoter DNA
 RNAP binding sites
 Operator – repressor binding
 Other TF binding sites
Start site of txn is +1

Initiation
 RNA polymerase
 4 core subunits
 Sigma factor (σ)–
determines promoter
specificity
 Core + σ = holoenzyme
 Binds promoter sequence
 Catalyzes “open complex” and
transcription of DNA to RNA

RNAP binds specific promoter
sequences
 Sigma factors recognize consensus

-10 and -35 sequences
RNA polymerase promoters
TTGACA TATAAT
Deviation from consensus -10 , -35 sequence leads to

weaker gene expression
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
 Most bacteria have major and alternate sigma factors
 Promote broad changes in gene expression
 E. coli 7 sigma factors
 B. subtilis 18 sigma factors
 Generally, bacteria that live in more varied

environments have more sigma factors
Sigma factors
Sigma subunit Type of gene controlled # of genes controlled
70 RpoD Growth/housekeeping ~1000
54 RpoN N2; stress response ~15

S RpoS Stationary phase, virulence ~100
S RpoH Heat shock ~40
F RpoF Flagella-chemotaxis ~40
32 RpoE Extreme ?heat shock, unfolded proteins ~5
FecI Ferric citrate transport ~5
E. coli can choose between 7 sigma factors and about 350

transcription factors to fine tune its transcriptional output
An Rev Micro Vol. 57: 441-466 T. M. Gruber
What regulates sigma factors
 Number of copies per cell (σ70 more than

alternate)
 Anti-sigma factors (bind/sequester sigma
factors)
 Levels of effector molecules
 Transcription factors
Bacterial RNAP numbers
 In log-phase E. coli:
 ~4000 genes
 ~2000 core RNA polymerase molecules
 ~2/3 (1300) are active at a time
 ~1/3 (650) can bind σ subunits.
 Competition of σ for core determines much of a cell’s

protein content.
Lac operon control
• Repressor binding prevents RNAP binding promoter
• An activating transcription factor found to be

required for full lac operon expression: CAP (or Crp)
lac operon – activator and
repressor
CAP = catabolite
activator protein
CRP = cAMP receptor

protein
Activating transcription factors
Crp dimer w/ DNA
 Helix-turn-helix
(HTH) bind major
groove
of DNA
 HTH one of many
TF motifs
Cofactor binding alters conformation
 Crp binds cAMP, induces allosteric
changes glucose glucose
cAMP cAMP
Crp Crp
lac operon
no mRNA mRNA
Cooperative binding of Crp and RNAP
Binds more stably than either protein alone

Enhancers
• activating regions not
necessarily close to RNAP
binding site
NtrC example:
• NtrC required for RNAP to
form open complex
• NtrC activated by P
• P NtrC binds DNA, forms loop

that folds back onto RNAP,
initiating transcription
• signature of sigma 54
DNA-protein interaction assays
 Electrophoretic mobility shift assay

(EMSA)
 DNase I Footprinting
 Chromatin immunoprecipitation (ChIP)

EMSA
Radiolabel promoter sequence
Incubate one sample with cell lysates or

purified protein and the other without
TF will bind promoter sequence
TF-bound probe
Run DNA-protein mixture on
polyacrylamide gel and visualize w/
audoradiography Free probe
EMSA
CovR DNA binding protein
Binds to cylE promoter
Recognition sequence ‘TATTTTAAT’
CovR PcylE
DNase I Footprinting
Method to determine where a protein binds a DNA sequence
DNase I footprint
1 -- DNA sequence ladder

2 -- DNA sequence ladder
3 -- No protein
4 -- (+) RNA polymerase
5 -- (+) lac repressor
ChIP
 Crosslink proteins
bound to DNA
 Immunoprecipitate
lysate for specific
transcription factor,
RNAP, etc
 Analyze DNA bound
to protein by PCR
Transcription
Initiation
Elongation
Termination
Processing
Capping
Splicing
Polyadenylation
Turnover
Translation
Protein processing
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
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
Rho-dependent termination
 Rho is hexameric protein
 70-80 base segment of RNA
wraps around
 Rho has ATPase activity,
moves along RNA until site
of RNAP, unwinds
DNA/RNA hybrid
 Termination seems to
depend on Rho’s ability to
“catch up” to RNAP
 No obvious sequence
similarities, relatively rare
Transcriptional attenuation
 Attenuator site = DNA sequence where RNAP chooses
between continuing transcription and termination
 trp operon
 4 RNA regions
for basepairing
 2 pairs w/ 1 or 3
 3 pairs w/ 2 or 4
 Concentration of
Trp-tRNATrp determines
fate of attenuation
 At high Trp conc,
transcription stops via
Rho-independent
Anti-termination
λ phage encode protein that prevents termination
Two Component Systems
Two Component Systems
 ‘Histidine kinase’ senses environmental

changes- autophosphorylates at conserved
histidine residue
 Response regulator is phosphorylated by
activated sensor kinase at conserved aspartate-
activates or represses transcription/function
 Way for bacteria to sense environmental
changes and alter gene expression
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
Quorum Sensing in Vibrio fischeri
• at high cell density, V. fischeri

express genes for bioluminescence
• LuxI produces autoinducer

acyl-homoserine lactone
• AHL diffuses outside of cell
• when AHL reaches critical

concentration, it binds LuxR
• activated LuxR bound AHL

activates transcription of
luminescence genes

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Transcription in Prokaryotes

Laboratory of Molecular Biology

DNA Environmental change

Turn gene(s) on/off

Many places for control

 Multiple genes transcribed

 No nucleus, so transcription and

 Contain ribose instead of deoxyribose

 RNA more like proteins than DNA:

 RNAP binds, melts DNA

 Ribosomal RNA (rRNA) – form the core of

 Transfer RNA (tRNA) – adaptors that link

 Small regulatory RNA – also called non-

 RNA polymerase α α β β’σ

 RNAP binding sites

 Operator – repressor binding

 Other TF binding sites

Start site of txn is +1

 Sigma factor (σ)–

 Binds promoter sequence

 Catalyzes “open complex” and

transcription of DNA to RNA

 Sigma factors recognize consensus

Deviation from consensus -10 , -35 sequence leads to

 B. subtilis 18 sigma factors

 Generally, bacteria that live in more varied

Sigma subunit Type of gene controlled # of genes controlled

70 RpoD Growth/housekeeping ~1000

54 RpoN N2; stress response ~15

FecI Ferric citrate transport ~5

E. coli can choose between 7 sigma factors and about 350

 Number of copies per cell (σ70 more than

 ~2000 core RNA polymerase molecules

 ~2/3 (1300) are active at a time

 ~1/3 (650) can bind σ subunits.

 Competition of σ for core determines much of a cell’s

• Repressor binding prevents RNAP binding promoter

• An activating transcription factor found to be

CRP = cAMP receptor

Binds more stably than either protein alone

• P NtrC binds DNA, forms loop

 Electrophoretic mobility shift assay

 Chromatin immunoprecipitation (ChIP)

Incubate one sample with cell lysates or

TF will bind promoter sequence

1 -- DNA sequence ladder

 Bacteria need to end transcription at the

• Termination sequence has 2 features:

 ‘Histidine kinase’ senses environmental

• at high cell density, V. fischeri

• LuxI produces autoinducer

• AHL diffuses outside of cell

• when AHL reaches critical

• activated LuxR bound AHL

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