Transcript Extensive and global regulation of transcription Shifts in

Riboswitches
• Region in mRNA, usually the 5’ UTR, that
binds a ligand and affects expression.
• The ligand is usually a small molecule,
e.g., flavin mononucleotide (FMN).
• The portion that directly binds the ligand is
the aptamer.
• Expression can be affected transcriptionally
or post-transcriptionally.
Fig. 7.34
A riboswitch from the B. subtilis ribD operon that binds
FMN and promotes transcription termination.
Fig. 7.35
REGULON
•
Collection of genes that are not in the same operon
but are co-regulated
•
Example:
Maltose (mal) regulon: genes needed to metabolize
maltose (glucose-glucose)
• involves multiple promoters and operons
• Activated by:
– MalT (a protein that requires the inducer,
maltotriose)
– and CAP for some promoters
Extensive and global regulation of
transcription in prokaryotes
1. Regulated transcription during sporulation
in Bacillus subtilis
2. Circadian regulation of global transcription
in Synechococcus, a cyanobacterium
2 forms of Bacillus
Vegetative cells
(Growing and dividing)
Fig. 8.3
Mother cell forming endospore
(Dormant stage or cell)
endospore
Spore resistant to heat and stress, and can turn back into a vegetative cell.
Endospore formation
1. Occurs in certain species of soil bacteria.
2. Triggered by lack of nutrients.
3. Requires turning off of many vegetative
genes, and turning on of spore-specific
genes.
4. Requires 3 sigma factors (σ29, σ30 and
σ32 or σE, σH and σC) in addition to the
vegetative sigma factor (σ43 or σA).
Similar to Fig. 8.5
σA
σE
Specific transcription in
vitro by σA and σE.
The in vitro-synthesized (with 32PUTP) RNA was hybridized to Southern
(DNA) blots of the above DNA
digested with the indicated restriction
enzymes.
Conclusion: The σA RNAP initiates only at the
Veg. promoter, but the σE RNAP initiates at the
veg. and sporulation promoters
Fig. 8.5
The function of the putative
sporulation-specific gene in
the previous experiment was
unknown.
So, transcription of a wellcharacterized sporulation
gene was performed with 4
different RNAPs, each with a
different sigma (σA, σB, σC,
and σE ).
Only σE transcribed the spoDII
promoter.
Fig. 8.6
What about genes that need to be expressed at high
levels at more than one stage in development?
One mechanism is to have 2 promoters:
Example: The spoVG gene of B. subtilis has σE and
σB promoters.
Fig. 8.8
Some sigma factors are, themselves,
sporulation-specific genes.
• Sigma K is the product of 2 sporulation
genes, spoIVCB and spoIIIC
- recombination forms the gene
- only happens in mother cell during spore
formation; the endospore remains
unrecombined
Circadian Rhythms
1. Oscillate with a period of ~24 hours
2. Phase determined by light-dark cycle
3. Once entrained, continue in “constant
conditions”
4. Show temperature compensation
Circadian
Bioluminescence
Rhythm in
Gonyaulax
(a eukaryote)
A natural rhythm
Temp. compensation
http://www.ucmp.berkeley.edu/history/linnaeus.html
Input
Output
Light
Development
Physiology
Behavior
Clock
Gene expression
An engineered circadian rhythm of
bioluminescence in Synechoccocus.
PpsbAI - promoter for psbAI gene
luxA + luxB = bacterial (Vibrio) luciferase
How many genes in Synechococcus
are circadian regulated?
•
Kondo et al. used promoter tagging
approach:
1. Transform promoterless luxA-luxB gene
fusion into Synechococcus so that it
integrates randomly - when it integrates
downstream of a promoter, get a bioluminescent
transformant.
2. Screen transformants for bioluminescence.
3. Determine how many show circadian rhythm
of bioluminescence.
Promoterless DNA construct
used for transforming
Synechococcus
Mid-day
Bioluminescent colonies that
are tracked with a computer
Night-time controlled imaging system –
track ~100 colonies at a time.
Results & Conclusion
1. Of ~30,000 transformants, ~800 had high
levels of bioluminescence.
2. Of the 800, all showed circadian rhythm of
bioluminescence.
3. Circadian rhythms of different phases and
amplitudes were observed.
Conclusion: The transcription of most genes in
Synechococcus are regulated by the
circadian clock (in addition to their other
modes of regulation).
What is the Clock?
- Regulatory proteins that form an
autofeedback loop!
Kai A,B,C genes in Synechococcus
Transcription in Organelles
endosymbiosis
α–Proteobacterium–like
organism
Mitochondrion
endosymbiosis
Cyanobacterium-like organism
Chloroplast
What about transcription of these highly evolved
and derived genomes?
Mitochondrion from Bean root tip
Mito. from transfer cell
Mitochondrial RNA Polymerase: A
phage-like RNAP
1.
2.
3.
4.
5.
Core enzyme: 1-subunit, phage-like enzyme.
Specificity factor needed to initiate at promoter
- human POLRMT enzyme needs two factors
- factors A (TFAM) and B (TFBM)
Similar core enzyme in animal, fungal and plant
mitochondria, but different specificity factors.
Promoter is usually a 9-10 bp sequence.
Genes usually encoded in the nucleus.
Evolutionary questions about the
Mitochondrial RNA Polymerase
• How and when was the phage-like RNAP
acquired?
• What happened to the bacterial RNAP (Rpo)
genes in the original endosymbiont?
Look at earlier eukaryotes
• Pylaiella, a eukaryotic
brown alga
• Phage-like polymerase
gene in the mitochondrial
genome
– suggests the nuclearencoded RNAP came
from the endosymbiont
a.k.a. “Mung”
• Reclinomonas, primitive
eukaryote
• Bacterial RNAP (rpo)
genes in mitochrondrial
genome (pseudogenes?)
• Suggests the
endosymbiont also had
rpo genes.
During evolution, phage-like polymerase was
transferred to the nucleus (from the
endosymbiont), and the rpo genes were lost.