Ch. 14

Regulatory proteins have two binding sites:

-One for a small effector molecule -The other for DNA

The lac operon can be transcriptionally regulated via...

1. By a repressor protein 2. By an activator protein

Gene regulation is important for cellular processes such as..

1. Metabolism 2. Response to environmental stress 3. Cell division -Regulation can occur at any of the points on the pathway to gene expression

To investigate this phenomenon, Jacob and Monod focused their attention on lactose metabolism in E. coli. Key experimental observations that led to an understanding

1. The exposure of bacterial cells to lactose increased the levels of lactose-utilizing enzymes by 1000- to 10,000-fold. 2. Antibody and labeling techniques revealed that the increase in the activity of these enzymes was due to the increased synthesis of the enzymes. 3. The removal of lactose from the environment caused an abrupt termination in the synthesis of the enzymes. 4. Mutations that prevented the synthesis of a particular protein involved in lactose utilization showed that a separate gene encoded each protein.

There are two distinct transcriptional units:

1. the actual lac operon: DNA elements and structural genes 2. the lacl gene

feedback inhibition

A method of metabolic control in which the end product of a metabolic pathway acts as an inhibitor of an enzyme within that pathway. -A common mechanism to regulate the activity of metabolic enzymes -if the concentration of the final product becomes high It will bind to enzyme 1 Thereby inhibiting its ability to convert substrate 1 into intermediate 1

Riboswitches

Folded RNAs that act as switches regulating protein synthesis in response to environmental conditions -First discovered in 2001 •RNA exists in two different secondary conformations -One is active -The other inhibits gene expression -Conversion between forms is due to the binding of a small molecule -3-5% of all bacterial genes may be regulated by riboswitches

(c) Repressor protein, corepressor molecule, repressible gene

In the absence of a corepressor, this repressor protein will not bind to the DNA. Therefore, transcription can occur. When the corepressor is bound to the repressor protein, this causes a conformational change that allows the protein to bind to the DNA and inhibit transcription.

(a) Repressor protein, inducer molecule, inducible gene

In the absence of the inducer, this repressor protein blocks transcription. The presence of the inducer causes a conformational change that inhibits the ability of the repressor protein to bind to the DNA. Transcription proceeds.

allolactose

Inducer of the lac operon; isomer of lactose generated by b-galactosidase -acts as a small effector molecule to regulate the lac operon

Genetic regulatory proteins that respond to small effector molecules have two functional domains.

One domain is a site where the protein binds to the DNA; the other domain is the binding site for the effector molecule.

repressed operon

RNA polymerase cannot bind to the promoter site and the genes will not be transcribed due to either a corepressor or inhibitor

anabolic=repressjble

The inhibitor or corepressor is the small molecule that is the product of the operon (ex, tryptophan in trp biosynthesis)

catabolic=inducible

The substance to be broken down (or a related compound) acts as the inducer (ex. allolactose from lactose)

effector molecule function:

They function in two ways Bind activators and cause them to bind to DNA Bind repressors and prevent them from binding to DNA Genes that are regulated in this manner are termed inducible ex inducer, co-repressor, or inhibitor

(d) Activator protein, inhibitor molecule, repressible gene

This activator protein will bind to the DNA without the aid of an effector molecule. The presence of an inhibitor causes a conformational change that releases the activator protein from the DNA. This inhibits transcription.

operon

a group of two or more genes under the transcriptional control of a single promoter -encodes a polycistronic RNA that contains the coding sequence for two or more structural genes -One biological advantage of an operon organization is that it allows a bacterium to coordinately regulate a group of two or more genes that are involved with a common functional goal; the expression of the genes occurs as a single unit. An operon contains several important DNA sequences Promoter; terminator; structural genes; operator

inducible, negative control mechanism

allolactose (inducer) binds to the lac repressor and inactivates it

lactose present

alolactose binds to repressor and inactivates the repressor protein so that transcription can occur -inducible operon, allolactose present

polycistronic RNA

an RNA that contains the sequences for two or more genes

A second way to regulate translation is via the synthesis of..

antisense RNA An RNA strand that is complementary to mRNA

cap site

binds the catabolite activator protein (CAP)

operator

binds the lac repressor protein

inhibitor

binds to an activator protein and prevents it from binding to the DNA. Both corepressors and inhibitors act to reduce the rate of transcription. Therefore, the genes they regulate are termed repressible genes. -a small effector molecule may inhibit transcription by binding to the activator

micF RNA

binds to the 5' region of the ompF mRNA, inhibiting ribosome binding and therefore translation -This prevents the cell from accumulating too high a concentration of ions -encodes iRNA, not protein -also known as the antisense RNA

lac operon

contains a CAP site; promoter (lacP); operator site (lacO); three structural genes, lacZ, lacY, and lacA; and a terminator. LacZ encodes the enzyme β-galactosidase, an enzyme that cleaves lactose into galactose and glucose. As a side reaction, β-galactosidase also converts a small percentage of lactose into allolactose, a structurally similar sugar

benefit of regulating genes..

encoded proteins will be produced only when required

lacZ

encodes B-galactosidase -Enzymatically cleaves lactose and lactose analogues Also converts lactose to allolactose (an isomer)

trpL

encodes a short peptide called the leader peptide which functions in attenuation In the case of the trp operon, transcription terminates shortly past the trpL region -Thus attenuation inhibits the further production of tryptophan -The segment of trp operon immediately downstream from the operator site plays a critical role in attenuation -The first gene in the trp operon is trpL

lacA gene

encodes galactoside transacetylase, an enzyme that covalently modifies lactose and lactose analogs -functional necessity remains unclear

lacY gene

encodes lactose permease, a membrane protein required for the active transport of lactose and analogues into the cytoplasm of the bacterium

effector molecules

exerts its effects by binding to an activator or repressor. The binding of the effector molecule causes a conformational change in the regulatory protein and thereby influences whether or not the protein can bind to the DNA -bind to regulatory proteins but not to DNA directly -in some cases, the presence of a small effector molecule may increase transcription ie inducer

The most common way to regulate gene expression in bacteria is by...

influencing the initiation of transcription -The rate of RNA synthesis can be increased or decreased

In the presence of glucose, the enzyme adenylyl cyclase is...

inhibited This decreases the levels of cAMP (inducer) in the cell Therefore, cAMP is no longer available to bind CAP (activator)k Transcription rate decreases

transcriptional regulation

involves the actions of regulatory proteins that can bind to the DNA and affect the rate of transcription of one or more nearby genes

repressor

is a regulatory protein that binds to the DNA and inhibits transcription -Transcriptional regulation by a repressor protein is termed negative control

activator

is a regulatory protein that increases the rate of transcription -regulation by an activator protein is considered to be positive control

inducer

is a small effector molecule that causes transcription to increase. An inducer may accomplish this in two ways: It could bind to a repressor protein and prevent it from binding to the DNA, or it could bind to an activator protein and cause it to bind to the DNA -Genes that are regulated in this manner are called inducible genes.

corepressor

is a small effector molecule that binds to a repressor protein, thereby causing the protein to bind to the DNA

no lactose

lacZYA off; repressor blocks RNA polymerase & prevents transcription -repressed operon, no allolactose

OmpF (outer membrane protein F)

larger porin protein dominant when E. coli is in dilute environment allows more diffusion of solutes OmpF protein is preferentially produced at low osmolarity At high osmolarity its synthesis is decreased Via the anti-sense strategy

small effector molecule in catabolite repression is....

not glucose This form of regulation involves a small molecule, cyclic AMP (cAMP) It is produced from ATP via the enzyme adenylyl cyclase cAMP binds an activator protein known as the Catabolite Activator Protein (CAP) which binds to the CAP site enabling transcription to occur -low transcription levels without it

regulatory proteins

proteins involved in the expression of control genes

translational regulatory protein

recognizes sequences within the mRNA In most cases, these proteins act to inhibit translation These are known as translational repressors

enzyme adaptation

refers to the observation that a particular enzyme appears within a living cell only after the cell has been exposed to the substrate for that enzyme -François Jacob and Jacques Monod at the Pasteur Institute in Paris were interested in this phenomenon They focused their attention on lactose metabolism in E. coli

TPP (thiamin pyrophosphate) riboswitch

regulates translation of the thi operon polycistronic mRNA (encodes enzymes needed for TPP biosynthesis) Binding of TPP to this mRNA allows the formation of stem-loop that sequesters the Shine-Dalgarno sequence......... Translation is blocked

Transcriptional regulation involves the actions of two main types of regulatory proteins:

repressor and acitvator

trpR gene

repressor gene that codes for repressor protein that binds to abundant tryptophan and bind to trp operon operator and turn off transcription

promoter

signals the beginning of transcription by binding RNA polymerase

terminator

specifies the end of transcription

inducible operon

structural genes are not transcribed unless an inducer is present

attenuation

the decrease or loss of virulence can occur in bacteria because transcription and translation are coupled During attenuation, transcription actually begins but it is terminated before the entire mRNA is made an early termination of transcription via the formation of a terminator 3-4 stem-loop, causing RNA pol to pause and allowing the U-rich sequence to dissociate from the DNA...remember rho-independent termination of transcription Conditions that favor the formation of the 3-4 stem-loop rely on the translation through the trpL mRNA (when tryp levels are high)

gene regulation

the level of gene expression can vary under different conditions

In bacteria, the most common way to regulate gene expression is by influencing

the rate at which transcription is initiated -At the level of transcription, this means that the rate of RNA synthesis can be increased or decreased

diauxic growth

the sequential use of two sugars by a bacterium

Genetic regulation in bacteria is exercised predominantly at the level of..

transcription, however can occur at translation or posttranslation

trp operon

tryptophan binds to the repressor protein and enables it to repress gene transcription. -involved in the biosynthesis of the amino acid tryptophan repressible operon The genes trpE, trpD, trpC, trpB and trpA encode enzymes involved in tryptophan biosynthesis The genes trpR and trpL are involved in regulation of the trp operon

lacl gene

Not considered part of the lac operon Has its own promoter, the i promoter Constitutively expressed at fairly low levels Encodes the lac repressor The lac repressor protein functions as a tetramer Only a small amount of protein is needed to repress the lac operon

cAMP-CAP complex

binding of cAMP to CAP. binds to CAP site. increases rate of transcription. -an example of transcriptional regulation that is inducible and under positive control

constitutive

-Genes that are unregulated -They have essentially constant levels of expression -Frequently, constitutive genes encode proteins that are continuously necessary for the survival of the organism

Catabolite repression of the lac operon

-lac operon is repressed when glucose and lactose are present -when glucose is used up, lactose inducible enzymes are then induced -transcription of lac operon requires both that lactose be present and that glucose not be present When exposed to both lactose and glucose E. coli uses glucose first, and catabolite repression prevents the use of lactose When glucose is depleted, catabolite repression is alleviated, and the lac operon is expressed The sequential use of two sugars by a bacterium is termed diauxic growth

translational repressors

A protein that blocks translation by binding to mRNAs and preventing their attachment to the ribosome. Translational repressors inhibit translation in one of two ways 1. Binding next to the Shine-Dalgarno sequence and/or the start codon This will sterically hinder the ribosome from initiating translation 2. Binding outside the Shine-Dalgarno/start codon region They stabilize an mRNA secondary structure that prevents initiation

Posttranslational Regulation

A second strategy to control the function of proteins is by the covalent modification of their structure.. Affect protein function Some modifications are irreversible Proteolytic processing Attachment of prosthetic groups, sugars, or lipids Other modifications are reversible and transiently affect protein function Phosphorylation (-PO4) Acetylation (-COCH3) Methylation (-CH3

(b) Activator protein, inducer molecule, inducible gene

This activator protein cannot bind to the DNA unless an inducer is present. When the inducer is bound to the activator protein, this enables the activator protein to bind to the DNA and activate transcription.