AP Biology Molecular Genetics

1. AP BiologyMolecular Genetics Regulation of Gene Expression in Prokaryotes Chapters 18 – Section 18.1, pages 351-356

2. First, some review: • One Gene-One Enzyme Hypothesis • Beadle & Tatum identified genes as being responsible for the production of enzymes • Central Dogma of Molecular Genetics

3. The need for Regulation • Cells have hundreds of genes, but will not need all of those proteins all of the time. • The enzymes a cell needs at any given time will be a function of the environment and conditions it is exposed to • In a multicellular organism, each cell has identical genetic material but produces only the proteins it needs to function. • Differentiation of cells into a variety of tissues results from variations in gene activity • For example, muscle cells will express the genes for actin and myosin, but not for hemoglobin or insulin.

4. The Operon Hypothesis • Sounds like a Jason Bourne movie, but actually a theory about control of gene activity • In bacteria many genes have an “operator” region downstream of the promoter which affects transcription • The promoter is the region that binds the RNA polymerase • The operator region can bind a “repressor” protein. The repressor protein blocks the movement of the polymerase • If the action of the polymerase is blocked, transcription is shut down (“repressed’)

5. Negative vs. Positive Control Negative Control • The gene is turned on unless the repressor binds to the operator • Since the repressor turns off the operon, control is negative Positive Control • The gene is on, and transcription occurs • If an activator protein binds, it will facilitate the binding of the polymerase, increasing the rate of transcription

7. The trp operon • Tryptophan is a necessary nutrient. The common intestinal bacterium E. coli will use tryptophan when it is available from its environment, but will need to produce tryptophan when it isn’t available. • E. coli makes tryptophan in a 3 step metabolic pathway from precursor molecules. The pathway requires 3 enzymes. • The genes for the enzymes are adjacent to each other on the chromosome, and are transcribed in a single mRNA with 5 start codons and 5 stop codons • The operon is regulated by a single promoter and operator • The genes will be transcribed unless tryptophan is present • When tryptophan is present it binds to the repressor and changes its shape, allowing it to bind to the operator region to turn the gene off

8. The trp operon

9. Repressible Negative Control • The gene is generally turned on, but can be repressed (turned off) by the binding of a repressor protein • The repressor protein is present in the cell, but in an inactive form. The repressor will become activated by the binding of a corepressor molecule • The corepressor may be the end product of the enzymatic reaction that the operon codes for. • In the case of the trp operon, tryptophan is the end product of the metabolic pathway and also the corepressor. • When the cell has an abundance of tryptophan, the cell will shut off the mechanism for making more tryptophan

10. Repressible negative control

11. Trp Feedback Inhibition

12. The trp Pathway • The tryptophan pathway consists of 3 enzymatic reactions • The end product, tryptophan, is produced from an organic precursor with 2 intermediates • The 3 enzymes are made from polypeptides coded for on 5 genes transcribed on a single mRNA

13. The trp Pathway • The first enzyme in the pathway has an allosteric site • The end product, tryptophan, can bind to the allosteric site of enzyme 1 and inhibit its activity (“allosteric inhibition”)

14. Inducible Negative Control • Inducible = able to be turned on • Negative = controlled by the binding of a repressor • This type of control is characteristic of genes that are usually not needed. The repressor protein is usually bound to the operator, blocking transcription • If an inducer molecule binds to the repressor it will detach from the operator, turning the gene on

15. The lac pathway • E. coli can digest lactose (a disaccharide) into the 2 simple sugars glucose and galactose. • Once digested, the simple sugars are metabolized through normal pathways • The process involves 3 enzymes, all transcribed from one operon on a single mRNA • There is no real advantage to digesting lactose if the cell already has glucose available. It’s more economical to metabolize glucose till the cell runs out, then switch to lactose metabolism • But there is no point to making enzymes for lactose digestion if there is no lactose available

16. The lac operon • The lac operon is an example of inducible negative control. The gene is turned off unless the bacteria have lactose available as a food source. • The inducer molecule is an isomer of lactose (called allolactose). When lactose is present the operon becomes activated

17. The lac operon

18. The lac operon - Details

19. Lactose or Glucose? • Glucose is easier for E. coli to metabolize than lactose, so if both foods are available the cells will use the glucose first. • The lac genes will be turned on, but transcribed at a low rate • The rate will be increase by the binding of an activator protein (positive control) only when glucose is scarce • When glucose is plentiful, the cells metabolic rate will be high. The cells content of ATP will be high, and the cell will have very little AMP. The content of AMP will determine the rate of transcription. The binding of AMP to the activator protein changes its shape, allowing it to bind to the the operator region and facilitate the activity of the polymerase

20. The role of AMP, ADP, and ATP • The energy currency of the cell is ATP. It is produced by phosphorylating ADP, much as ADP is produced by phosphorylating AMP • The mechanisms for phosphorylation were discussed in detail in our Bioenergetics unit, but for the sake of review: • Substrate level phosphorylation (in glycolysis) • Chemiosmotic phosphorylation (in the electron transport chain) • A cell with an abundance of food will have produced a large quantity of ATP. This means that the quantity of AMP and ADP will be low since almost all of it will have been phosphorylated • A cell with a high content of AMP is starving, because it has used up its available ATP and hasn’t been able to make more

21. cAMP • When the cell has a large quantity of AMP (again, the cell must be starving because its ATP is used up) some of that AMP is modified. Technically, the phosphate at the 5’ position forms a second bond to the pentose at the 3’ position – basically a phosphodiester bond self contained within a single nucleotide. Because this creates a ring structure the AMP is now “cyclic” AMP (cAMP) • Don’t get too hung up on the details. cAMP serves as a signaling molecule, and the signal it’s sending is starvation. cAMP means the cell is hungry, and because it’s hungry it needs to turn on some alternative pathways to metabolism

22. Positive Control

23. Gene Regulation Videos • Gene Regulation • http://www.youtube.com/watch?v=3S3ZOmleAj0&list=PLFCE4D99C4124A27A • Alternative Approaches to Molecular Biology • https://www.youtube.com/watch?v=TnpCMgtDPgk&list=PLF83B8D8C87426E44 • The lac operon • https://www.youtube.com/watch?v=2TL8rY9Rc_A&list=PLF83B8D8C87426E44

24. Rationale • The central dogma of molecular genetics states that genes are molecules of that must be transcribed into molecules of which then are translated into • The process of transcription is directed by an enzyme called • Translation is directed by particles of RNA and protein called which bind to the • Molecules of then align to codons, matching the appropriate to the code • The ribosome then catalyzes the formation of bonds between the forming a polypeptide

25. Regulation • Economy demands that genes are only active when the cell need the proteins that they code for. If there is an accumulation of the end product of an enzymatic pathway, it benefits the cell to turn the gene (on/off) • It is most economical to exert control at the level of (transcription/translation)by blocking the activity of the(ribosomes/mRNA/tRNA/RNA polymerase)because the cell will not need to bear the expense of producing (ribosomes/mRNA/tRNA/RNA polymerase)

26. Fundamentals • The promoter region is the area where binds, allowing • The operator region overlaps the promoter on the downstream end, acting as a binding site for a repressor protein. Binding of the repressor (allows/prevents)binding of the RNA polymerase, (preventing/promoting) transcription • Operons that use repressor proteins are under (positive/negative)control because the repressor turns the gene (on/off)

27. Repressible vs Inducible The repressor protein is active when it binds to the operator. Some repressors are already the proper shape to bind actively, others only become active when also bound to an allosteric molecule. • An (inducer/corepressor)will (activate/inactivate)the repressor, causing it to attach to the operator region, turning the gene (on/off) • An (inducer/corepressor)will (activate/inactivate)the repressor, causing it to detach from the operator region, turning the gene (on/off)

28. Feedback systems • Negative feedback occurs when an accumulation of a molecule (often the end product of a pathway) stimulates the pathway to become (more/less) active • The result of negative feedback is (amplification/homeostasis) • An accumulation of tryptophan (increases/decreases) the need for more tryptophan. The tryptophan will act as (an inducer/a corepressor), binding to a repressor protein making the repressor (active/inactive) • The repressor will then (attach to/detach from)the operator region, turning the operon (on/off)by (allowing/blocking)transcription

29. Trp pathway feedback loop • Tryptophan also exerts negative feedback control directly on the metabolic pathway. Excess tryptophan will bind to the allosteric site of the first enzyme in the trp synthesis pathway. The trp acts as an allosteric (activator/inhibitor), changing the shape of the enzyme to make it become (capable/incapable) of binding to the substrate • Exerting control at the enzyme level allows (faster/slower)response and is (easier/more difficult)to reverse once tryptophan levels diminish

30. Identify all parts of the system

31. The lac Operon • E. coli can use either glucose (a monosaccharide) or lactose (a disaccharide) for energy. If both food sources are available the cell benefits by using the (glucose/lactose) first because it doesn’t need to be digested. • If lactose is available, the lac operon needs to be turned (on/off)by (attaching/detaching) the repressor protein. This is accomplished by binding allolactose to the repressor. The allolactose acts as (an inducer/a corepressor) • If glucose is available glucose will be metabolized, resulting in an increase in the amount of (AMP/ATP) and a decrease in the amount of (AMP/ATP) • A “hungry” cell will have (high/low) quantities of cAMP which will bind to the Activator Protein (increasing/decreasing)the rate of transcription for the lac operon

32. Identify all parts of the system

33. Concept Check 18.1, page 356 • How does the binding of the trpcorepressor and the lac inducer to their respective repressor proteins alter repressor function and transcription in each case? • Describe the binding of RNA polymerase, repressors, and activators to the lac operon when both lactose and glucose are scarce. What is the effect of these scarcities on the transcription of the lac operon? • A certain mutation in E. coli changes the lac operator so that the active repressor cannot bind. How would this affect the cell’s production of b-galactosidase? (b-galactosidase is enzyme 1 of the lac pathway)