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Riboswitches

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Title: Riboswitches


1
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.

2
Fig. 7.34
A riboswitch from the B. subtilis ribD operon
that binds FMN and promotes transcription
termination.
3
Fig. 7.35
4
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

5
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

6
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.
7
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 (s29, s30 and s32 or sE,
    sH and sC) in addition to the vegetative sigma
    factor (s43 or sA).

8
Similar to Fig. 8.5
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 above
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. 8.5
9
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. 8.6
10
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 sE and sB promoters.
Fig. 8.8
11
  • 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

12
(No Transcript)
13
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

14
CircadianBioluminescence Rhythm in Gonyaulax(a
eukaryote) A natural rhythm
Temp. compensation
15
http//www.ucmp.berkeley.edu/history/linnaeus.html
16
A Circadian System
Gene expression
17
An engineered circadian rhythm of bioluminescence
in Synechoccocus.
PpsbAI - promoter for psbAI gene luxA luxB
bacterial (Vibrio) luciferase
18
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 -
    when it integrates downstream of a promoter, get
    a bioluminescent transformant.
  • Screen transformants for bioluminescence.
  • Determine how many show circadian rhythm of
    bioluminescence.

19
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
20
Results Conclusion
  • 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 regulated by the circadian
    clock (in addition to their other modes of
    regulation).

21
What is the Clock?
- Regulatory proteins that form an autofeedback
loop! Kai A,B,C genes in Synechococcus
22
Transcription in Organelles
endosymbiosis
aProteobacteriumlike organism
Mitochondrion
endosymbiosis
Cyanobacterium-like organism
Chloroplast
What about transcription of these highly evolved
and derived genomes?
23
?Mitochondrion from Bean root tip
Mito. from transfer cell?
24
Mitochondrial RNA Polymerase A phage-like RNAP
  • 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.

25
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?

26
Look at earlier eukaryotes
  • Pylaiella, a eukaryotic brown alga
  • Phage-like polymerase gene in the mitochondrial
    genome
  • suggests the nuclear-encoded RNAP came from the
    endosymbiont

a.k.a. Mung
27
  • 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.
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