1 Genetic regulation Genotype is not phenotype: bacteria possess many genes that they are not using at any particular time. Transcription and translation are expensive; why spend ATP to make an enzyme you don’t need? Operon –Genes physically adjacent regulated together Regulon –Genes dispersed but controlled by same proteins –Operator sequences must be same/similar

2 More on Regulation Two important patterns of regulation: Induction and repression. –In induction, the genes are off until they are needed. –In repression, the genes normally in use are shut off when no longer needed. Negative control –Binding of protein to the DNA prevents transcription Positive control –Binding of protein to DNA promotes transcription

3 Repressible operons Operon codes for enzymes that make a needed amino acid (for example); genes are “on”. –Repressor protein is NOT attached to DNA –Transcription of genes for enzymes needed to make amino acid is occurring. The change: amino acid is now available in the culture medium. Enzymes normally needed for making it are no longer needed. –Amino acid, now abundant in cell, binds to repressor protein which changes shape, causing it to BIND to operator region of DNA. Transcription is stopped. This is also Negative regulation (protein + DNA = off).

4 Repression picture Transcription by RNA polymerase prevented.

5 Regulation can be fine tuned The more of the amino acid present in the cell, the more repressor-amino acid complex is formed; the more likely that transcription will be prevented.

6 Structure of the Lac operon KEY: P O are the promoter and operator regions. lac Z is the gene for beta-galactosidase. lac Y is the gene for the permease. lac A is the gene for a transacetylase. lac I, on a different part of the DNA, codes for the lac repressor, the protein which can bind to the operator.

7 Binding of small molecules to proteins causes them to change shape Characteristic of many DNA-binding proteins Regulation of operons: Inducible operons: Repressor protein comes off DNA Repressible operons: Repressor protein attaches to DNA

8 How the lac operon works When lactose is NOT present, the cell does not need the enzymes. The lac repressor, a protein coded for by the lac I gene, binds to the DNA at the operator, preventing transcription. When lactose is present, and the enzymes for using it are needed, lactose binds to the repressor protein, causing it to change shape and come off the operator, allowing RNA polymerase to find the promoter and transcribe.

9 Lactose is not actually the inducer Low basal levels of beta- galactosidase exist in the cell. This converts some lactose to the related allolactose which binds to the lac repressor protein. Synthetic inducers such as IPTG with a similar structure can take the place of lactose/allolactose for research purposes.

10 Glucose is the preferred carbon source

Positive regulation Presence of lactose is not enough –In diauxic growth graph, lactose is present from the start. Why isn’t operon induced? Presence of glucose prevents positive regulation –NOT the same as inhibiting –Active Cyclic AMP receptor protein (CRP) needed to bind to DNA to turn ON lactose operon (and others) –Presence of glucose (preferred carbon source) prevents activation of CRP activator-protein

Additional controls Attenuation –Seen w/ repressible operons, fine tuning –Ribosome does not stall, transcription terminated mRNA rapidly degraded –Signal “to make” stops, residual mRNA destroyed Examples of –Antisense RNA: binds to mRNA, prevents use –DNA rearrangements; genes flip in place, different gene product produced –Ribosome binding protein prevents translation 12

Global control: modulons Different operons/regulons affected by same environmental signal –Presence of glucose –Change from O 2 to anaerobic growth –Nitrogen limitation; phosphate starvation –Growth rate control –Cell division –Stationary phase; entering starvation state One method of control: alternate sigma factors –Sigma controls which promoters are used 13