RNAP, sigma factors, and transcription

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RNAP, sigma factors, and transcription

• 10-19-2009

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Transcription machinery

• RNA Polymerase (RNAP)
• Core enzyme (E)
• Homo-dimer of a subunit (rpoA)-
• b (rpoB) and b (rpoC) subunits for RNAP
• w subunit (rpoZ)
• s70 (rpoD) to recognize promoter sequence and
  carry out the vast majority of transcription

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RNAP composition in three domains
Werner (2007) Mol Microbiol
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Structural evolution of RNAP
Archaea
Eucaryote
Bacteria

• Eukaryotic RNAPs have evolved from an
  archaeal-like ancestral RNAP by the simple
  addition of motifs, domains and subunits, whereas
  the differences between the bacterial and
  archaeal RNAP include changes to the core
  structure.
• Werner (2008) Trends in Microbiology

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Transcription

• A cyclic process of DNA dependent RNA
  polymerization (transcripts)
• Template recognition
• Initiation
• Abortive transcription (lt 9 bp)
• RNAP-DNA closed complex formation? melting of 2S
  DNA ? open complex formation? release of s factor
• Elongation
• transcription pausing (50 bp)
• Termination

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Transcription Initiation
Closed complex (holo-enzyme)
Open complex
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Bacterial Promoter
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Promoter recognition
- Primary s subunits share 4 conserved regions -
The regions 2.4, 3.0, and 4.2 have been
implicated in specific recognition of the -10,
the extended -10 (TG), and the -35 elements
Nature Rev Micro (June 2008)
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Signals turn on alternative sigma factors

• The major sigma factor Es70
• Alternative sigma factors
• s32 (sH ) controls heat shock promoters
• s54 (sN ) control promoters for nitrogen
  assimilation
• s38 (sS) turns on stationary-phase promoters
• sF for flagellum-related functions
• sFecI for promoters involved in iron transport
• sE controls response to extracytoplasmic
  stresses
• 
  J Bacteriol (2006) 1884589-

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RpoS- the central regulator of the general
protective response
sS-controlled genes

• In Salmonella enterica and Escherichia coli,
  unfavorable growth conditions (including nutrient
  limitation, outright starvation, low temperature,
  osmotic shock, as well as other stresses)
  initiate a generalized stress response
• In association with RNA polymerase, EsS directs
  transcription of as much as 10 of the E. coli
  genome, including genes necessary for stress
  resistance and virulence

JB(2005) 187 1591-
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sB- stress resistance sigma factor in G()
bacteria
sB-dependent promoter

• Analysis of conservation of the sB regulon genes
  revealed that only 3 conserved genes in all 4
  bacteria? the sB regulon has evolved to perform
  niche-specific functions

JB 2007, 189 43844390
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Virulence associated sigma factors
G()
G (-)
Conservation of sB Regulon (mostly encoding
pathogenic traits) in Low GC G() Bacteria
JB 2007 Jun
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Alternative sigma factors and virulence
extracytoplasmic function sigma factors
Micro Mol Biol Rev (2005) 69527-
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The ECF s factors of B. subtilis

• Regulatory overlap and functional redundancy in
  drug resistance

JB 2007, p. 69196927
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Heat shock response sigma factor (sH)

• The heat shock response (HSR) is classically
  defined as the cellular response to temperature
  increase via upregulation of a set of heat shock
  proteins by a transcription factor, Escherichia
  coli s 32
• Following upshift to temperatures of gt 37C but
  within the growth range of the organism
• 
  Micro and Mol Biol Rev (2008) 72545-

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Acid stress activation (sE stress response) in
Salmonella enterica serovar Typhimurium

• sE is activated by unfolded outer membrane
  proteins (OMPs OmpC)
• can also be activated by acid stress, which is
  independent of the unfolded OMP signal or the
  DegS protease

Molecular Microbiology (2009)
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PDZ domain

• A common structural domain of 80-90 amino-acids
  found in the signaling proteins of bacteria,
  yeast, plants, viruses, and animals
• An acronym combining the first letters of three
  proteins post synaptic density protein (PSD95),
  Drosophila disc large tumor suppressor (DlgA),
  and zonula occludens-1 protein (zo-1)
• also referred to as DHR (Dlg homologous region)
  or GLGF (glycine-leucine-glycine-phenylalaine)
  domains

From Wikipedia, the free encyclopedia
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Multiple sigma factors in a genome

JB 2008, Jan ECF-extracytoplasmic function sigma
factors SigC, SigD, SigE, SigG, SigH, SigI, SigJ,
SigK, SigL, SigM in Mycobacterium tuberculosis
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Regulation of sigma factors

• A reversible interaction of s and a protein
  inhibitor (an anti-s factor)
• Bacillus sB factor
• controls the expression of more than 60 proteins
  in response to different conditions
• Regulated by anti-sB via partner-switching

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Partner switching regulation
RsbW An anti-sigma factor and kinase
kinase
Serine Phosphatase
RsbV An anti-anti-sigma factor
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No sS homologues in Alphaproteobacteria- Sigma
factor mimicry involved in regulationof general
stress response

• PhyR contains a sigma factor-like domain of the
  extracytoplasmic function subfamily linked to a
  receiver domain of a response regulator
• PhyR does not act as a genuine sigma factor but
  instead controls gene expression indirectly
  through protein-protein interactions
• In response to a stress, PhyR is phosphorylated
  and interacts with its anti-sigma factor, NepR

PNAS (2009) Online
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Transcription factors

• DNA binding domain
• However, many transcription actors bind directly
  to RNAP without binding to DNA by affecting
  specific kinetic steps on the pathway to open
  complex formation, thereby regulating RNA output
  from specific promoters.
• Regulatory domain

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Molecular mechanism of transcription

Micro Mol Bio Rev, Sept. 2009, p. 481509
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DNA binding transcription factors

• FIS (factor for inversion stimulation protein)
  and HNS (histone-like nucleoid structuring
  protein) function in nucleoid organization as
  well as in gene regulation
• FIS protein binds to the site near the major
  rpoS promoter during exponential growth,
  resulting in repression of rpoS transcription

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H-NS as a gene silencer

• H-NS protein in Salmonella Typhimurium has a key
  role in selectively silencing the transcription
  of large numbers of horizontally acquired AT-rich
  genes, including those that make up its major
  pathogenicity islands

Nature Reviews Microbiology online 27 December
2006
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Anti-silencing

• integration of the horizontally acquired genes
  into the existing gene regulatory networks

Microbiology (2008), 154, 25332545
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FIS and HNS control sigma factor selection

• Regulation of Dps which is a nucleoid-associated
  protein and plays a major role in condensation
  of the E. coli chromosome in stationary phase
• FIS present in fast-growing exponential culture
  traps the s70-RNA polymerase at the dps promoter
• H-NS contributes to the regulation of sigma
  factor selection at low temperature. It
  selectively prevents binding of s70-RNA polymerase

Prevent hypercondensation
Molecular Microbiology (2008) 68
13451347
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Abortive initiation

• Backtracking of RNAP
• RNAP moves backward along the template reversing
  the translocation steps in case of some failure
  of the RNA through chemical damage or
  misincorporation
• Association of Gre proteins help to cleave the
  RNA and restart new RNA synthesis
• To increase transcription fidelity

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• Elongation complex-
  Release of sigma factor

• After synthesis of 912 nt of RNA? loss of
  RNAP-DNA contacts? dissociation of s? formation
  of an elongation complex (transcription bubble)

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Transcription elongation

• Movement of transcription bubble
• Compression (RNAP-27 bp DNA) and expansion
  (RNAP-35 bp DNA)- an inchworm movement
• The movement is not uniform and inevitable
• Control factors
• Anti-termination factors
• Nus proteins
• Transcription disruption factors
• Gre and Mfd proteins

Annu. Rev. Microbiol. 2008. 62211
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Anti-termination factors
Detailed genetic analysis of l phage N-mediated
anti-termination - The essential elongation
proteins NusA and NusG, - the anti-terminator
binds at a specified site and forms a persistent
complex with RNAP that causes it to bypass
terminators
Ribosomal protein S10
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RNAP-NusA structure

• The resulting structure formed between the RNA
  polymerase and NusA from Bacillus subtilis
  provides important insights into the transition
  from an initiation complex to an elongation
  complex, and how NusA is able to modulate
  transcription elongation and termination.

EMBO report published online 14 August 2009
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Intrinsic transcription terminator

• A specific structure consisting of a hairpin
  followed by poly U residues at the 3terminus of
  the RNA
• 90 of E. coli genome sequence encode protein,
  the remaining non-coding sequence is densely
  packed with regulatory signals for transcription
  initiation and termination

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Intrinsic termination The intrinsic terminator
has a GC-rich hairpin that forms in the emerging
transcript An adjacent U-rich segment about
nine nucleotides upstream of the RNA release site
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Rho-dependent termination In addition to
intrinsic termination, the remaining half of the
transcription termination rely on Rho protein to
bind the emerging transcript to pull it out of
the transcription complex
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Rho protein

• An essential hexameric ATP-dependent RNA
  helicase
• recognizes no specific consensus but rather binds
  to naked, un-translated RNA, favoring C-rich
  sites that contain little secondary structure
• Rho-dependent termination sites occur frequently
  in operons

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Rho strongly repressed transcription of
horizontal acquired genes
Rho specific inhibition by treatment with the
antibiotic bicyclomycin (BCM) Science (2008)
May 16
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Reduced-genome E. coli is resistant to Rho
inhibition
Rac prophage fragment
MDS42 genome has been reduced by targeted
deletion of prophage, IS, and K-island
Science (2008) May 16
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NusA and NusG support Rho termination to suppress
the toxic activity of foreign genes
Science (2008) May 16
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Mfd (mutation frequency decline) protein

• Function in transcription-coupled DNA repair
• An ATP-dependent DNA translocase to induce
  forward translocation of RNAP (remove stopped
  transcription complexes) and recruit the DNA
  excision repair machinery to the site?
  transcription pausing