Extensive and global regulation of transcription in prokaryotes

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Extensive and global regulation of transcription
in prokaryotes

• Regulated transcription during sporulation in
  Bacillus subtilis
• 2. Circadian regulation of global transcription
  in Synechococcus, a cyanobacterium

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2 forms of Bacillus
Vegetative cells (Growing and dividing)
Fig. 8.5
Mother cell forming endospore (Dormant stage or
cell)
endospore
Spore resistant to heat and stress, and can turn
back into a vegetative cell.
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Endospore formation

• Occurs in certain species of soil bacteria.
• Triggered by lack of nutrients.
• Requires turning off of many vegetative genes and
  turning on of spore-specific genes.
• Requires 3 sigma factors (s29, s30 and s32 or sE,
  sH and sC) in addition to the vegetative sigma
  factor (s43 or sA).

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Fig. 8.6
sA sE
Specific transcription in vitro by sA and sE.
The in vitro synthesized (with 32P-UTP) RNA was
hybridized to Southern (DNA) blots of the DNA
digested with the indicated restriction enzymes.
Conclusion The sA RNAP initiates only at the
Veg. promoter, but the sE RNAP initiates at the
veg. and sporulation promoters
Fig. 8.7
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The function of the putative sporulation-specific
gene in the previous experiment was unknown. So,
transcription of a well-characterized sporulation
gene was performed with 4 different RNAPs, each
with a different sigma (sA, sB, sC, and sE
). Only sE transcribed the spoDII promoter.
Fig. 8.8
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Some sigma factors are, themselves,
sporulation-specific genes. An interesting one
is sigma K, which is the product of 2 sporulation
genes, spoIVCB and spoIIIC. Recombination
between these 2 genes is necessary to create the
sigma K protein gene.
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Recombination to form sigma K only happens in the
mother cell genome during spore formation the
endospore genome remains unrecombined.
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What about genes that need to be expressed at
high levels at more than one stage in
development. One mechanism bacterial cells use
to do this is to have 2 promoters for the gene
Example The spoVG gene of B. subtilis has sE
and sB promoters.
Fig. 8.12
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Circadian Rhythms

• Oscillate with a period of 24 hours
• Phase determined by light-dark cycle
• Once entrained, continue in constant conditions
• Show temperature compensation

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CircadianBioluminescence Rhythm in GonyaulaxA
natural rhythm
Temp. compensation
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A Circadian System
Gene expression
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An engineered circadian rhythm of bioluminescence
in Synechoccocus.
PpsbAI - promoter for psbAI gene luxA luxB
bacterial (Vibrio) luciferase
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How many genes in Synechococcus are circadian
regulated?

• Kondo et al. used promoter tagging approach
• Transform promoterless luxA-luxB gene fusion into
  Synechococcus so that it integrates randomly.
• Screen transformants for bioluminescence.
• Determine how many show circadian rhythm of
  bioluminescence.

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Promoterless DNA construct used for transforming
Synechococcus
Mid-day
Bioluminescent colonies that are tracked with a
computer controlled imaging system track 100
colonies at a time.
Night-time
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Results Conclusions

• Of 30,000 transformants, 800 had high levels of
  bioluminescence.
• Of the 800, all showed circadian rhythm of
  bioluminescence.
• Circadian rhythms of different phases and
  amplitudes were observed.
• Conclusion The transcription of most genes in
  Synechococcus are clock-regulated, in addition to
  their other modes of regulation.

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What is the Clock?
- Regulatory proteins that form an autofeedback
loop! Examples KaiA,B,C Synechococcus Toc1
Arabidopsis Period (per) Drosophila Frequen
cy (frq) Neurospora Per timeless (tim) mouse
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References for Circadian rhythms

• Review article Dunlap, J., 1999, Cell
  96271-290
• Synechococcus papers
• Kondo et al., 1993, Proc. Natl. Acad. Sci USA
  90 5672-5676
• Liu et al., 1995, Genes Development 9
  1469-1478
• Kondo et al., 1994, Science 2661233-1236