Regulation of Gene Expression in Bacteria and Their Viruses Chapter 11

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

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.

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

Regulatory Proteins Control Transcription

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

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

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.

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

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

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

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.

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

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.

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

The lac operon is transcribe only in the presence of lactose

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

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

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?)

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

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

Operators are cis-acting Repressors are trans-acting

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

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

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

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?

The lac operon is transcribed only in the presence of lactose

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)

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.

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

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.

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.

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

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.