Bacterial Keys to Success Respond quickly to environmental changes –Simultaneous transcription and translation Avoid wasteful activities by using biochemical and genetic controls –Feedback inhibition of key enzymes –DNA binding proteins to control transcription Exchange genes with other organisms to improve adaptation to environment –Conjugation; exchange of plasmids and chromosomal DNA 1

Using DNA is expensive to the cell Model: protein of 50,000 daltons –Approximately 500 amino acids, so 500 codons 500 codons is 1500 RNA bases Transcription –Every base added (NTP to NMP) spends 2 phosphates, so equivalent of 2 ATP –1500 bases/mRNA = 3000 ATP Translation –1 ATP to move ribosome, 1 to charge tRNA /codon –2 ATP/ codon = 2 * 500 = 1000 ATP for every single protein molecule produced. 2

Biosynthesis is expensive 3 acids_clip_image004_0001.gif

Feedback Inhibition The first enzyme in the pathway has an allosteric site –The first substrate in the pathway fits into the active site –The end product fits into the allosteric site Shape change from allosteric binding distorts active site –First step in pathway is inhibited –More efficient than preventing a later step 4

5 Biochemical regulation: allosteric enzymes Allo = other; steric = space. Many enzymes not only have an active site, but an allosteric site. Binding of a molecule there causes a shape change in the enzyme. This affects its function.

6 Feedback inhibition of pathways

Genetic Regulation Biochemical regulation –Prevents the synthesis of unneeded small molecules Genetic regulation –Prevents the synthesis of the enzymes that make the small molecules. –Results in additional energy savings Different view of genotype vs. phenotype –Can the bacterium metabolism the sugar sucrose? (genotype) –Is the bacterium presently able to metabolize it? (phenotype) 7

8 Organization of genes Many genes work together –Code for proteins that work in same pathway –Systems control genes together, increasing or decreasing their transcription as needed by the cell. Operon –Genes physically adjacent and regulated together Regulon –Genes dispersed but controlled by same proteins

9 How Gene activity is controlled Characteristic of many DNA-binding proteins Shape change may lead to increased binding to DNA or to decreased binding depending on protein. Binding to DNA affects amount of transcription. Binding of small molecules to proteins causes them to change shape

Operon Structure 10 The Promoter is the site on DNA recognized by RNA polymerase as place to begin transcription. Operator is location where regulatory proteins bind. Promoter and Operator are defined by function.

11 Patterns of Regulation Control of transcription is through binding of proteins to the DNA Negative control –Binding of protein to the DNA prevents transcription Positive control –Binding of protein to DNA promotes transcription 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.

Binding of lac repressor to DNA 12 More lac Repressor Research - Lewis & Lu Labs

13 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).

14 Repression picture Transcription by RNA polymerase prevented.

15 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.

Induction Genes normally off are turned on –Example of negative regulation Presence of inducer allows gene expression –Inducer binds to repressor which comes off DNA 16

17 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.

18 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.

19 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.

20 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

Function of CRP 22 Low glucose: Adenylate cyclase active cAMP abundant cAMP binds to CRP Complex binds to DNA Promotes transcription