RNA Structure

1
Lecture 7

• RNA Structure
• and
• Prokaryotic Transcription

2
RNA Structure

• Contain ribose instead
• of deoxyribose
• 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 become
functional
3
RNA synthesis

• RNAP binds, melts
• DNA into open form
• nucleosides added
• 5 ? 3
• so bottom strand
• is really template

4
RNA Structure Types of RNA

• Messenger RNA (mRNA) genes that encode proteins
• Ribosomal RNA (rRNA) form the core of ribosomes
• Transfer RNA (tRNA) the adaptors that link
  amino acids to mRNA during translation
• Small nuclear RNA (snRNA) RNA splicing of
  pre-mRNA to mRNA
• Small regulatory RNA also called non-coding RNA

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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

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Types of RNA
Today, focus on mRNA Catalytic RNAs will come
later
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Transcription information transfer
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Transcription information transfer

• Regulons and Stimulons

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Martinez-Antonio, J. et al 2003. 6(3)482-9
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Prokaryotic Transcription

• bacteria have operons
• groups of related genes
• w/ same promoter that are transcribed
  polycistronically
• polycistronic RNA multiple genes
  transcribed as ONE TRANSCRIPT
• no nucleus, so transcription and translation can
  occur simultaneously

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Eukaryotic Transcription

• no operons groups of related genes can be on
  different chromosomes
• each gene has its own
• RNA transcript
• (monocistronic)
• transcription and translation separated by
• nucleus and RNA processing

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Transcriptional Control

• Very important to
• be able to express genes when they are needed
• Be able to repress genes when they are
  detrimental
• not waste energy expressing genes when they are
  not needed

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Transcriptional Control
Many places for control to occur

Transcription Initiation Elongation Termination P
rocessing Capping Splicing Polyadenylation Turnove
r Translation Protein processing
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Transcriptional Control
Transcription Initiation Elongation Termination P
rocessing Capping Splicing Polyadenylation Turnove
r Translation Protein processing
Control of initiation usually most important.
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Initiation

• RNA polymerase
• Transcription factors
• Promoter DNA
• RNAP binding sites
• Operator repressor binding
• Other TF binding sites
• start site of txn is 1

a a ßßs
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Initiation

• RNA polymerase
• 4 core subunits
• Sigma factor (s)
• determines promoter
• specificity
• Core s holoenzyme
• Binds promoter sequence
• Catalyzes open complex and transcription
• of DNA to RNA

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Initiation lac operon
CAP catabolite activator protein
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RNAP binds specific promoter sequences

• Consensus sequences
• -10 and -35
• Sigma factors recognize -10 and -35 sequences

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RNA polymerase promoters
TTGACAT
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
• Most bacteria have major sigma factor and
  alternate sigma factors
• Promotes 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
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Sigma factors
Extreme heat shock/extracytoplasmic
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
22
What regulates sigma factors

• Number of copies per cell (s70 more than
  alternate)
• Anti-sigma factors (bind/sequester sigmas)
• Levels of effector molecules
• Transcription factors

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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 s subunits.
• 1200 s subunits.
• Competition of s for core determines much of a
  cells protein content.

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Footprint of lac operon control region

• Repressor binding prevents RNAP binding promoter
• An activating transcription factor found to be
• required for full lac operon expression CAP
  (or Crp)

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Bacterial transcription factors
lac operator
Many TFs bind inverted repeats as dimers More
interactions greater affinity
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Activating transcription factors
Crp dimer w/ DNA

• Helix-turn-helix (HTH) bind major groove
• of DNA
• HTH one of many
• TF motifs

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Cofactor binding alters conformation

• Crp binds cAMP, induces allosteric changes

glucose
cAMP
Crp
mRNA
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Cooperative binding of Crp and RNAP
Binds more stably than either protein alone
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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

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DNA-protein interaction assays

• Footprinting
• Electrophoretic mobility shift assay (EMSA)
• aka gel shift

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DNase I Footprinting
Method to determine where a protein binds a DNA
sequence
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Footprint of RNAP and lac repressor
1 -- DNA sequence ladder 2 -- DNA sequence
ladder 3 -- No protein 4 -- () RNA polymerase 5
-- () lac repressor
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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
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EMSA
Mandin, P., et al. (2005) Mol Micro 57 (5),
13671380.
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Transcriptional Control
Transcription Initiation Elongation Termination P
rocessing Capping Splicing Polyadenylation Turnove
r Translation Protein processing
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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 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 Rhos ability to
  catch up to RNAP
• No obvious sequence similarities, relatively rare

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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

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Antitermination

• ? phage encode protein
• that prevents
• termination
• E. coli contain
• several factors
• that work together
• for antitermination

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Transcription information transfer
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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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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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Two Component Regulatory Systems

• Sensor protein sense environmental changes
• Response regulator phosphorylated by sensor and
  does function (e.g. activate transcription)
• Phosphorelay cascade
• Way for bacteria to sense environmental changes
  and alter gene expression accordingly

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Two Component Regulatory Systems
Response regulator
P
P
P
P
Transcription
Promoter
regulon genes
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Salmonella and PhoP/PhoQ
SPI-1
SPI-2
Entry
Survival in vacuole
SsrB/SpiR
PhoP/PhoQ
SpiC
Survival genes
SPI-2 genes
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PhoP/PhoQ
SsrB/SpiR