1. Regulation of Gene Expression in Bacteria and Their Viruses
Chapter 11

2. Terms to get familiar with outside of class
Activator
Protein when bound to a cis acting regulatory DNA element, such as a promoter or an enhancer, activates transcription of an adjacent promoter
Repressor
Protein that binds to a cis-acting regulatory DNA element and prevents transcription
Operators
DNA element at one end of an operon that is the binding site for the repressor
Allosteric site
Site on a protein that binds a small molecule causing a change in the conformation of the protein that modifies the activity of its active site
Allosteric effector
Small molecule that binds to the allosteric site
Allosteric transition
Change for one conformation of a protein to another
Induction
Relief of repression of a gene or set of genes under negative control
Inducers
Environmental agent that triggers transcription from an operon

3. Gene Regulation in prokaryotes
Bacteria must regulate genes to survive!
Nutritional opportunists
They must be able to recognize environmental conditions in which they should activate or repress the transcription of the relevant genes
They must be able to toggle on or off, like a switch, the transcription of each specific gene or group of genes.

4. Prokaryotic transcriptional regulation: Genetic Switches
Promoters must be recognized for transcription to start
However, binding sites near the promoter can serve as activators or repressors of transcription
Binding sites for repressors are operators in bacteria
Binding site for activators are activator binding sites
The activators or repressors that bind to DNA elements constitute genetic switches that control the efficiency and abundance of gene expression

5. Regulatory Proteins Control Transcription

6. Activators and repressors
Both activators and repressors proteins must be able to recognize when environmental conditions are appropriate for their actions
Therefore they must be able to exist in two states:
When it can bind DNA
When it can’t bind DNA

7. Cheat sheet for positive and negative regulation AND Inducible vs repressible operons
Positive vs Negative Regulation

8. A researcher is studying a genetic switch in a new species of bacteria
A researcher is studying a genetic switch in a new species of bacteria. She notices that, in order for a gene of interest to be transcribed, an activator must be bound to a region upstream of the promoter. This is an example of
negative regulation.
positive regulation.
neutral regulation.
misregulation.

9. Regulatory Proteins Sites
DNA binding domain
Location where the protein binds DNA
Allosteric site
Acts as a sensor that sets the DNA binding domain in one of two modes: functional or nonfunctional
Allosteric effectors are small molecules that bind to the allosteric site with specificity and alter the proteins activity
Allosteric transition – change of shape due to binding of the effector

10. Lac regulatory circuit (lac operon)
The lac genes (3 structural genes total)
Metabolism of lactose requires two enzymes
Permease (gene Y) to transport lactose into the cell
β-galactosidase (gene Z) to modify lactose into allolactose and cleave the lactose molecule to yield glucose and galactose
Transacetylase (gene A) which is not needed for lactose metabolism
Transfers an acetyl group from acetyl-CoA to β-galactosides. Its precise function as part of the lac operon is not understood.
All three are made from one mRNA
Coordinately controlled - genes whose transcription is controlled by a common means
Polycistronic message

11. Role of -galactosidase
14 β -D-glucopyranose
16 β-D-glucopyranose

12. Lets Review the Regulatory components of the lac system
The gene for Lac repressor (Gene I)
The lac promoter site (P)
Location where RNA pol binds to initiate transcription
Lac Operon – consists of P, O, Z, Y and A
(lac) I is not part of the operon
Operon is defined as a segment of DNA that encodes a multigene mRNA as well as an adjacent common promoter and regulatory region.

13. Repressor protein controls the lac operon
Simplified lac operon model
Coordinate expression of the Z, Y, and A genes is under the control of the product of I gene
When the inducer binds the repressor, the operon is fully expressed….induction

14. A graduate student has just ordered a large quantity of a small molecule from a company. The student will treat bacteria with this molecule, which will bind to a specific activator protein, thus changing its shape and resulting in transcription of a target gene. This small molecule is a(n)
a) activator. b) repressor. c) allosteric effector. d) promoter. e) operon.

15. A scientist is interested in gene transcription in E
A scientist is interested in gene transcription in E. coli, and she is studying the promoter and operator regions of a specific gene. When it comes to proteins, ____________ will bind to the promoter, and ___________ will bind to the operator of her gene of interest.
RNA polymerase; a repressor
a repressor; RNA polymerase
RNA polymerase; an activator
a repressor; an activator

16. The lac operon is transcribe only in the presence of lactose

17. Concept check
As I have told you, Lac Z, Y, and A genes are polycistronic… WHAT DOES THAT MEAN AGAIN?

18. To study gene regulation you need three things
A biochemical assay to measure mRNA or protein (or both)
Reliable conditions which the expression levels differ in wt genotypes
WHY would you want to alter gene expression?
Genetic mutation that perturbs the level of expression

19. Jacob and Monod work to provide evidence for the operator and repressor
Used a synthetic inducer because allolactose is hydrolyzed by -galactosidase and IPTG is not
IPTG still binds of repressor
Found many mutations that altered gene expression but to study the interactions, they needed diploids
Determine dominance
Created partial diploids by inserting F’ factors (WHAT ARE THESE AGAIN?)

20. Examine mutations that inactivate the structural genes for β-galactosidase and permease
Identifying Operator mutants
Constitutive mutations OC (always on regardless of the presence of lactose)
No IPTG IPTG

21. Examine mutations that inactivate the structural genes for β-galactosidase and permease
Identifying Repressor mutants

22. Operators are cis-acting
Repressors are trans-acting

23. Genetic evidence for allostery
The repressor contains a lactose-binding site
Is super-repressor mutation, prevents expression in the presence of the inducer

24. The super-repressor
The mutation is in the allosteric site which prevents binding

25. Genetic analysis of the lac promoter
RNA polymerase contacts the promoter at specific sequences
Mutations in the promoter are cis-acting

26. Molecular characterization of the Lac repressor and the lac operator
The operator is a specific DNA sequence
What would have more effect on Z and Y transcription… an Oc mutation or an Is mutation?

27. The lac operon is transcribed only in the presence of lactose

28. Lac system Evolved to maximize energy efficiency
Two environmental conditions have to be met for lactose metabolites to be expressed
Lactose needs to be present
Glucose cannot be present
The cell can capture more energy from the breakdown of glucose than other sugars
Glucose causes catabolite repression
Lactose breaks down into glucose and galactose, so the presence of glucose stops lac operon expression
Via a Catabolite activator protein (CAP)

29. Catabolite Repression of the lac Operon: Positive Control
Activator
Protein
The lac operon has an added level of control so that the operon is inactive in the presence of glucose even if a lactose is present. An allosteric effector, cAMP, binds to the activator CAP to permit the induction of the lac operon. However, high concentrations of glucose catabolites inhibit production of cAMP, thus failing to produce cAMP-CAP and thereby failing to activate the lac operon.

30. A summary of the lac operon
Negative and positive control of the lac operon

32. Transcription of the lac operon is maximally activated when
lactose is absent and glucose is present.
lactose is absent and glucose is absent.
lactose is present and glucose is present.
lactose is present and glucose is absent.

33. Dual Positive and Negative Control: The Arabinose Operon
Arabinose operon is an example in which a single DNA-binding protein may act as either a repressor or an activator Map of the ara operon I-inducer
an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde (CHO) functional group.

34. Review: Repression and activation compared
The Inducer binds to both the repressor and the activator

35. AraC serves as an activator and as a repressor
Operon transcription can be regulated by both activation and repression. Operons regulating the metabolism of similar compounds, such as sugars, can be regulated in quite different ways.