RiboswitchesSlide 2Slide 3REGULONExtensive and global regulation of transcription in prokaryotesSlide 6Endospore formationSlide 8Slide 9Slide 10Slide 11Slide 12Circadian RhythmsCircadian Bioluminescence Rhythm in Gonyaulax (a eukaryote) A natural rhythmSlide 15Slide 16Slide 17How many genes in Synechococcus are circadian regulated?Slide 19Results & ConclusionSlide 21Transcription in OrganellesSlide 23Mitochondrial RNA Polymerase: A phage-like RNAPEvolutionary questions about the Mitochondrial RNA PolymeraseLook at earlier eukaryotesSlide 27Riboswitches•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. 7.34A riboswitch from the B. subtilis ribD operon that binds FMN and promotes transcription termination.Fig. 7.35REGULON•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 promotersExtensive and global regulation of transcription in prokaryotes1. Regulated transcription during sporulation in Bacillus subtilis2. Circadian regulation of global transcription in Synechococcus, a cyanobacterium2 forms of BacillusVegetative cells(Growing and dividing)Mother cell forming endospore(Dormant stage or cell)endosporeSpore resistant to heat and stress, and can turn back into a vegetative cell.Fig. 8.3Endospore formation1. 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).Specific transcription in vitro by σA and σE.The in vitro-synthesized (with 32P-UTP) RNA was hybridized to Southern (DNA) blots of the above DNA digested with the indicated restriction enzymes. σA σEConclusion: The σA RNAP initiates only at the Veg. promoter, but the σE RNAP initiates at the veg. and sporulation promoters Fig. 8.5Similar to Fig. 8.5The 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 (σA, σB, σC, and σE ).Only σE transcribed the spoDII promoter.Fig. 8.6What 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. 8.8Some 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 unrecombinedCircadian Rhythms1. Oscillate with a period of ~24 hours2. Phase determined by light-dark cycle3. Once entrained, continue in “constant conditions”4. Show temperature compensationCircadianBioluminescence Rhythm in Gonyaulax(a eukaryote) A natural rhythmTemp. compensationhttp://www.ucmp.berkeley.edu/history/linnaeus.htmlClockLightInput OutputDevelopmentPhysiologyBehaviorGene expressionAn engineered circadian rhythm of bioluminescence in Synechoccocus.PpsbAI - promoter for psbAI geneluxA + luxB = bacterial (Vibrio) luciferaseHow 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 SynechococcusBioluminescent colonies that are tracked with a computer controlled imaging system – track ~100 colonies at a time. Mid-dayNight-timeResults & Conclusion1. 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 SynechococcusTranscription in Organellesα–Proteobacterium–like organism MitochondrionCyanobacterium-like organismChloroplastWhat about transcription of these highly evolved and derived genomes?endosymbiosisendosymbiosisMitochondrion from Bean root tip Mito. from transfer cellMitochondrial RNA Polymerase: A phage-like RNAP1. Core enzyme: 1-subunit, phage-like enzyme.2. Specificity factor needed to initiate at promoter- human POLRMT enzyme needs two factors - factors A (TFAM) and B (TFBM) 3. Similar core enzyme in animal, fungal and plant mitochondria, but different specificity factors.4. Promoter is usually a 9-10 bp sequence.5. 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 nuclear-encoded RNAP came from the endosymbionta.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