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 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) Mother cell forming endospore (Dormant stage or cell) endospore Spore resistant to heat and stress, and can turn back into a vegetative cell. Fig. 8.3

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 (σ 29, σ 30 and σ 32 or σ E, σ H and σ C ) in addition to the vegetative sigma factor (σ 43 or σ A ).

Specific transcription in vitro by σ A and σ E. The in vitro-synthesized (with 32 P- UTP) RNA was hybridized to Southern (DNA) blots of the above DNA digested with the indicated restriction enzymes. σ A σ E Conclusion: The σ A RNAP initiates only at the Veg. promoter, but the σ E RNAP initiates at the veg. and sporulation promoters Fig. 8.5 Similar to Fig. 8.5

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 (σ A, σ B, σ C, and σ E ). Only σ E 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 σ E and σ B 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

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 Bioluminescent colonies that are tracked with a computer controlled imaging system – track ~100 colonies at a time. Mid-day Night-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 α–Proteobacterium–like organism Mitochondrion Cyanobacterium-like organism Chloroplast What about transcription of these highly evolved and derived genomes? endosymbiosis

 Mitochondrion from Bean root tip Mito. from transfer cell 

Mitochondrial RNA Polymerase: A phage-like RNAP 1.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 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.