Bio 1107 Chapter 18

Mutant

Cell or individual with a mutation

AraC

Codes for the activator

Mechanisms of Regulation

Genes can be under: *Transcriptional control* *Translational control* *Post-translational control* *ALL three types of regulation occur in bacteria*

Global gene regulation

The coordinated regulation of many genes.

Review of Negative control of Transcription

The lac operon is transcribed efficiently when lactose is present and glucose is absent. The lac operon is under negative control A repressor protein binds to an operator sequence in DNA near the lac operon promoter to prevent transcription of lac operon genes. When lactose is present, it binds to the repressor and causes it to fall off the operator, allowing transcription to occur. Glucose inhibits transcription of the lac operon by inhibiting lactose transport into the cell, by preventing an activator protein from binding near the promoter, or through a combination of these mechanisms.

Regulation of CAP

When glucose levels outside the cell are low, CAP-cAMP complex stimulates RNA polymerase binding

Mutagens

X-rays, UV light, or chemicals that damage DNA and increase mutation rates

Regulon

*A set of separate genes or operons that contain the same regulatory sequences and that are controlled by a single type of regulatory protein*. Regulons allow bacteria to respond to challenges that include shortage of nutrients, sudden changes in temperature, exposure to radiation, or shifts in habit. Regulons can be under negative control by a repressor protein or positive control by an activator protein.

Post translational control

*After translation proteins must be activated by chemical modification*, such as the addition of a phosphate group, in order to function. Provides most rapid response because only one step is needed to activate an existing protein

How Does Glucose Regulate the lac Operon?

*Transcription of the lac operon is greatly reduced when glucose is present* --Even when lactose is also available --When glucose is already available, the cell does not need to produce more by cleaving lactose Two mechanisms for how glucose prevents lac operon expression: 1. *Inducer exclusion* 2. *Inhibition of an activator, called CAP*

Which is most efficient?

*Transcriptional* control is the most efficient --It controls gene expression before the cell expends many resources --However, it is the slowest For example, an enzyme isn't produced if regulatory proteins block RNA polymerase binding the promoter Some genes are transcribed *constitutively* (all the time): It is critical to realize that gene expression is not an all-or-none proposition. Genes are not just "on" or "off" - instead, *the level of expression can vary between these extremes*. DNA --> mRNA --> protein --> activated protein

Ara operon

Contains three genes that allow E.coli to use the sugar arabinose

Central dogma

DNA -> RNA (mRNA) -> protein

lac operon

The group of genes involved in lactose metabolism

Intro to control of gene expression

A cell does not express all of its genes all of the time. --Cells are very selective about --Which genes they express --In what amounts genes are expressed --When genes are expressed Regulation of gene expression is critical to cells

lacA gene

A fourth gene, lacA, is also part of the lac operon The lacA gene codes for the enzyme transacetylase Exports excess sugar from the cell

Colony

A large number of identical cells descended from a single cell

Operon

A set of coordinatley regulated bacterial genes that are transcribed together into one mRNA.

Allosteric regulation

A small molecule binds to a protein and causes it to change shape and activity. When the inducer binds to the repressor, the repressor can no longer bind to DNA, and transcription can proceed.

Inducer

A small molecule that triggers transcription of a specific gene

Medium

A substance on which cells can grow

Genetic screen

Any technique for identifying cells with certain types of mutations in a large population

Review of global gene regulation

Bacterial cells often need to coordinate the expression of large sets of genes in response to changing environments.

Constitutive mutants

Cells that are abnormal because they produce a product at all times

Review of gene regulation and information flow

Changes in gene expression allow bacterial cells to respond to environmental changes Most gene products are produced or activated only when needed Gene expression can be controlled at three levels: transcription, translation, or post-translation (protein activation). Transcriptional control can be negative or positive. Negative control occurs when a regulatory protein prevents transcription. Positive control occurs when a regulatory protein increases the frequency of initiating transcription.

Arabinose

Found in plant cell walls Without arabinose in the environment, the ara operon is not transcribed, but when arabinose is present, transcription of the ara operon is turned on by a protein called AraC

Replica plating

Method used to identify bacterial colonies that had certain mutations. 1. Bacterial colonies spread cells on a "master plate" containing a solid growth *medium* 2. Bacterial cells allowed to divide on the surface of the growth medium so that each cell produce a *colony* 3. Researchers then transferred each colony to a set of other plates (*replica plates*) and observed their growth under different conditions.

Metabolizing Lactose

Monod and Jacob studied E. coli lactose metabolism E. coli uses lactose only when glucose is depleted Before E. coli can use lactose 1. It must transport it into the cell with the protein galactoside permease 2. It must cleave it with the enzyme β-galactosidase to produce glucose and galactose E. coli produces high levels of β-galactosidase only when lactose is present in the environment --Lactose acts as an *inducer* because it is a molecule that triggers transcription of a specific gene If glucose is also present, β-galactosidase is not produced in high levels because glucose negatively regulates β-galactosidase expression *Significant amounts of β-galactosidase are produced only when lactose is present and glucose is absent*

Review of identifying regulated genes

Mutants that failed to cleave lactose, to transport lactose into the cell, or to regulate transcription of lac operon genes were isolated from many mutant cells that researchers generated and screened. Transcription may be constitutive or regulated. Constitutive expression occurs in genes whose products are required at all times, such as genes that encode glycolytic enzymes.

Negative control

Occurs when a regulatory protein called a *repressor* binds to DNA and shuts down transcription

Positive control

Occurs when a regulatory protein called an *activator* binds to DNA and triggers transcription

Positive control of Transcription

Positive control of transcription occurs when a protein called an activator binds to a regulatory sequence in DNA. Activator proteins bind to RNA polymerase in addition to DNA. Binding between the activator and RNA polymerase increases the rate of transcription initiation. The *ara operon* codes for genes required for metabolism of the sugar *arabinose*. The operon is controlled by the *AraC* regulatory protein. AraC is an activator when bound to arabinose and a repressor when the protein is not bound to arabinose.

Cotranscription

Results in the coordinated expression of three genes

Operator

Section of DNA in the lac operon

Transcriptional control

The cell can only make mRNAs for particular proteins. If genes for unneeded proteins are *not* transcribed into mRNA, then ribosomes cannot make these proteins. This form of control *occurs when regulatory proteins affect RNA Polymerase's ability to bind to a promoter and initiate transcription*. Slowest *"RNA is only made for some genes, due to regulatory proteins at the promoter"*

Translational control

The cell can prevent the mRNAs for unneeded proteins from being translated. Regulatory molecules can speed up mRNA degradation or ability of a mRNA to be translated can be affected. Faster than transcriptional because the mRNA is already made *"Not all RNAs are translated"* Control can occur through many mechanisms

Overview of gene regulation and information flow

The gut bacterium E. coli can use a wide array of carbohydrates to supply carbon and energy Control of gene expression allows E. coli to: --Respond to its environment --Switch its use of sugars Gene expression in bacteria was predicted to be triggered by specific signals from the environment

Replica plating in research of lacI

The initial mutant screens yielded three types of mutants. 1. Mutants were unable to cleave lactose - even when lactose was in the medium and transported into cells to induce the production of the β-galactosidase protein. 2. Cells produced β-galactosidase but failed to accumulate lactose inside the cell. 3. Mutants (most intriguing), could produce both β-galactosidase and galactoside permease, but had defects in regulating the expression of these products. To summarize observations: the normal product of the lacI gene prevents the transcription of lacZ and lacY when lactose is absent. Because lactose triggers the production of β-galactosidase, they proposed that the lacI gene or gene product interacts with lactose in some way. Lactose metabolism involved 3 genes: lacZ, lacY, and lacI. They concluded that lacZ and lacY code for proteins required for the metabolism and import of lactose, while lacI is responsible for some sort of regulatory function. When lactose is absent, the lacI gene or gene product shuts down the expression of lacZ and lacY. But when lactose is present, transcription of lacZ and lacY is induced. They discovered that the genes are close together. This was a crucial finding because it suggested that lacZ and lacY might be transcribed together.

Negative control of Transcription

The lacZ and lacY genes are under negative control --The lacI gene codes for a repressor --Binds on or near the lacZ and lacY promoter --Lactose induces transcription by removing the repressor LacI- mutants have no repressor --lacZ and lacY genes are expressed with or without lactose

Gene expression

The multistep process of converting information that is archived in DNA into molecules that actually do things in the cell. *Gene expression occurs when a protein of other gene product is synthesized and becomes an active cell*.

The operon model

Three hypotheses are central to the operon model: 1. The lacZ, lacY, and lacA genes are cotranscribed into one mRNA initiated from a single promoter ​2. lacI is a repressor of the lac operon --(a) lacI is expressed constitutively --(b) the repressor binds to the operator and --(c) blocks RNA polymerase holoenzyme from binding to the promoter 3. Lactose is the inducer that changes the shape of lacI, causing it to release from the operator --A type of allosteric regulation

Identifying regulated genes

To find mutants that are associated with a particular trait, the researchers had to complete 2 steps: 1. Generate a large number of cells with mutations at random locations on their genomes. Monod and Jacob did this by exposing E.coli populations to *mutagens* 2. Screen (genetic screening) the treated cells for mutants with defects in the process or biochemical pathway in question. In this case, defects in lactose metabolism.

Control of Transcription

Transcription can be regulated via: Negative control or Positive control.

Mechanism's of regulation

Transcriptional control, translational control, and post-translational control

Inducer exclusion

Transport of lactose into the cell is inhibited when glucose is high Lactose does not remove the repressor from the operator When glucose is low, more lactose enters, and the repressor is removed