Molecular Genetics Chapter 14

Components of lac operon

(1) Structural genes (a) lacZ gene (b) lacY gene (c) lacA gene (2) Regulatory region (a) Promoter region (lacP) (b) Operator region (lacO) (3) Repressor gene (lacI) (4) CAP site: Catabolite Activator Protein binding site

trpR gene

(not part of trp operon) produces repressor that binds to operator region - same as lac i

CAP is involved in:

- CAP facilitates RNA polymerase binding on the promoter -cooperative binding, enhances each other's binding character - CAP is also called cyclic AMP receptor protein

Life cycle of phage λ bacteriophage reproductive cycle

- Lytic cycle - lysogenic cycle

Feedback inhibition in posttranslational regulation:

- final product of the pathway inhibits activity of one or more enzymes in the pathway - enzyme within regulatory/allosteric site as well as catalytic site - binding of the final product to regulatory site changes conformation of enyzme - enzyme cannot bind to substrate

Genetic proofs to support operon model

- repressor gene produces a diffusible cellular product (proteins) - operator region is involved in regulation, but no gene product is known - operator region is adjacent to the structural genes to regulate transcription

Repressors in translational regulation:

- repressor recognition within mRNA - bind to Shine-Dalgarno sequence - binding may cause structural change on mRNA

How many promoters are in an operon? A)1 B)2 C)3 D)It depends on how many genes are present in the operon

A)1

An activator is present and results in the increase in transcription of the target gene. This is an example of _______________. A)termination B)positive control C)negative control D)feedback inhibition

B)positive control

CAP site

Catabolic Activator Protein binding site -DNA sequence recognized by catabolite activator protein - CAP facilitates RNA polymerase binding on the promoter (enhances gene expression) - CAP binding to CAP site is cAMP dependent

Cis-acting elements

DNA sequences in the vicinity of the structural gene that are required for gene expression

O1 site should be functional to repress:

O1 with O2 or O1 with O3

What are the three lac operon sites where lac repressor binds?

O3, O1, O2

Repressor must bind to ___ of the three operator sites to repress transcription.

Two

Regulatory proteins

action by direct binding to DNA - activators - repressors

CAP binding is dependent on ______.

cAMP

DNA that contains instructions for two or more structural genes produces monocistronic mRNA. ⊚true⊚false

⊚false

In the lac operon, the operator is an example of a trans-effect genetic regulation. ⊚true⊚false

⊚false

In the lac operon, the operator site is recognized by an activator protein. ⊚true⊚false

⊚false

Negative transcriptional regulation is conducted by activator proteins. ⊚true⊚false

⊚false

Operons that code for anabolic enzyme systems are typically regulated by inducers. ⊚true⊚false

⊚false

Operons that code for catabolic enzyme systems are typically regulated by repressors. ⊚true⊚false

⊚false

Constitutive genes are those that have constant levels of expression. ⊚true⊚false

⊚true

Repressor proteins are responsible for negative transcriptional regulation. ⊚true⊚false

⊚true

The form of regulation that involves a physical change in the shape of an enzyme is called allosteric regulation. ⊚true⊚false

⊚true

The regulation of the CAP complex using cAMP is an example of inducible genetic regulation. ⊚true⊚false

⊚true

The term enzyme adaptations used to describe an enzyme that appears in a living cell following exposure to a specific substrate. ⊚true⊚false

⊚true

Which of the following encode polycistronic mRNA? (Check all that apply.) A)Lac operon B)Operator site C)Trp operon D)CAP site

A)Lac operon C)Trp operon

Regulation of gene expression may occur at which of the following levels? (Check all that apply.) A)Post translation B)Constitutive expression C)Translation D)Transcription

A)Post translation C)Translation D)Transcription

Riboswitch

Binding of small molecule to a regulatory segment of mRNA causes conformational change and leads to modification of mRNA function - riboswitch of mRNA regulates its own translational activity and enzymatic activities - riboswitches are found in bacteria, archaea, algae, fungi, and plants

A gene is inducible and under negative control. Which of the following pairs will allow expression of this gene? A) Activator + repressor B) Activator + inhibitor C) Repressor + inducer D) Repressor + co-repressor

C) Repressor + inducer

A deletion in an operon removes the terminator. How will that affect the transcript that is produced from the operon? A)The transcript will be produced and normal in length B)The transcript will be produced, but shorter than normal C)The transcript will be produced, but longer than normal D)The transcript will produced, but will contain a deletion E)The transcript will not be produced

C)The transcript will be produced, but longer than normal

Allosteric regulation is accomplished by __________. A)a small molecule that fits into an enzyme's active site B)a large protein that blocks an enzyme's active site C)a small molecule that fits into a site on the enzyme that is not the active site D)a small molecule that covalently modifies a site on the enzyme that is not the active site

C)a small molecule that fits into a site on the enzyme that is not the active site

A deletion in an operon removes the promoter. How will that affect the transcript that is produced from the operon? A)The transcript will be produced and normal in length B)The transcript will be produced, but shorter than normal C)The transcript will be produced, but longer than normal D)The transcript will produced, but will contain a deletion E)The transcript will not be produced

E)The transcript will not be produced

Regulatory proteins involved in phage cycles

Following infection, two promoters are activated: PL and PR - produce 'cro protein' and 'N protein' - N protein has antitermination function (binds to RNA polymerase and prevents termination) Genes activated for lytic cycle - genes are initializing phage synthesis (O and P) - second antiterminator (Q) Genes activated for lysogenic cycle - integration for phage genome into bacterial chromosome (int) - excise gene (xis) - stabilizer for CII protein (cIII)

Possible stem-loop structures of trpL mRNA

No translation on mRNA from trpL gene (eukaryotes) - stable stem-loop formation of attenuator region - no transcription and translation of trp operon No tryptophan present - no charged trp-tRNA present - ribosome stops at trp codons - no stem-loop formation of attenuator region - continuous transcription and translation (tryptophan is produced) Tryptophan present - charged trp-tRNA present - ribosome moves until meets termination codon - stem-loop formation of attenuator region - transcription to trp operon is halted (no tryptophan produced)

Glucose and lactose absent

OFF (CAP and repressor bound)

Glucose present and lactose absent

OFF (repressor bound)

Glucose and lactose present

OFF/(ON but low expression) - CAP and repressor off

Glucose absent and lactose present

ON (CAP bound)

Antisense RNA in translational regulation:

RNA that binds to the complementary RNA or DNA sequence - complementary to mRNA

trp operon

Repressible operon - encodes enzymes involved with biosynthesis of tryptophan (amino acid)

Organization of trp operon

Structural genes: - TrpE, D, C, B, and A gene - transcribes polycistronic mRNA - Genes produce tryptophan synthetase

Repressors form a ______ and _____ _____.

Tetramer, DNA loop

Transcription regulation by riboswitches

Thaimine pyrophosphate (TPP) is an essential coenzyme for the functioning of various enzymes - transcriptional control of thi operon of B subtilis - when TPP is low, transcription is completed - when TPP is high, transcription is stopped

Types of riboswitches

Transcription - formation of rho-independent transcription termination hairpins Translation - inaccessible Shine-Dalgarno sequence (ribosome binding site) RNA stability - formation of self-degradable ribozyme Splicing - conformational change and splicing variant formation

Translation regulation by riboswitches

Translational control of thiMD operon of E. Coli - the thiMD operon encodes two enzymes involved with TPP biosynthesis - when TPP levels are low, translation is completed - when TPP levels are high, no translation

Lytic cycle may be induce by __ exposure.

UV - increases transcription of recA

Constitutive gene

a gene that is transcribed at a relatively constant level regardless of the cell's environmental conditions example: housekeeping genes (HSP90, b-actin)

Operon

a unit of genetic material that functions in a coordinated manner by a single promotor - contains an operator, a promotor, and structural genes - produces a polycistronic mRNA

Positive regulation

activator protein promotes transcription

High tryptophan levels:

active trp repressor - repression occurs - tryptophan is a corepressor or attenuation occurs (prokaryotes) - RNA is transcribed only to the attenuator sequence - stem-loop formation of attenuator sequence causes premature termination of transcription - first two trp codons control ribosomal movement by availability of charged trp-tRNA

cAMP is produced from _____ _____.

adenylyl cyclase

CAP-cAMP complex

binds to the CAP-binding site in the promoter region - enhances transcription - occurs when glucose is absent

λ regulation

cII protein is easily degraded by E. coli cellular proteases - these proteases are abundant if the bacterial growth conditions is favorable - if cII is degraded, λ repressor is not made and the lytic cycle is favored

OR region

contains three operators - acts as a genetic switch between lytic and lysogenic cycles - cro and λ repressor can bind to all three sites - these proteins determine the switch between the lytic and lysogenic cycles - during lysogenic cycle, λ repressor controls the switch - during lytic cycle, cro protein controls the switch

Effector proteins

do not bind directly to DNA, but react with repressors or activators (DNA binding proteins) - effectors cause conformational change in activator or repressor - Inducers, corepressors, and inhibitors

Corepressors

effector molecules that decrease gene expression by binding to repressors

Inducers

effector molecules that increase gene transcription by binding to activators or repressors - genes regulated by inducers are called 'inducible genes'

Inhibitors

effectors that bind to the activator and prevent it from binding to DNA - genes regulated by inhibitors are called 'repressible genes'

lacZ gene

encodes b-galactosidase (converts lactose to glucose and galactose)

trpL gene

encodes leader peptide (14 amino acids) - mRNA determines transcription and translation of trp operon

lacY gene

encodes permease (facilitates entry of lactose into bacterial cell; cotransports H+)

lacA

encodes transacetylase (removes toxic by-products from lactose digestion)

Catabolic repression/CAP inducible system

energy efficient system - glucose is a catabolite of lactose digestion

Posttranslational regulation

feedback inhibition and covalent modification of enzyme

Inducible regulation

gene expression is induced by presence of particular substance (inducer)

Dual control of lac operon

glucose inhibits adenylyl cyclase activity

Low tryptophan levels:

inactive trp repressor, transcription of the entire trp operon occurs

Activators

increase transcription causing positive control - may be naturally active or inactive

Catabolic genes in an operon are usually regulated in an ______ manner.

inducible

lac operon regulation

inducible and negative control system - transcription occurs when the repressor fails to bind to the operator region - gene is off by default

lac operon

inducible operon - encodes enzymes for lactose digestion - mRNA contains sequences for multiple proteins/enzymes

Repressors

inhibit the transcription process causing negative control - may be naturally active or inactive

Lactose present

lactose (allolactose) binds to repressor - binding leads to the conformational change in repressor - repressor cannot bind to operator region -then, the genes expressed

Gene regulation

level of gene expression can vary under different conditions - may occur at any stage of gene expression

Mutation on operator region (O-)

mutation blocks the passage of RNA polymerase -genes are always turned off

Genome of phage λ

normal λ phage genome is linear - linear genome turned into circular when it is injected into the host bacteria - various group of genes are responsible for lytic and lysogenic cycles lytic cycle: genes needed for new viral particle formation are expressed lysogenic cycle: integrase is expressed

Covalent modification of enzyme in posttranslational regulation:

permanent modifications - proteolytic processing (protein is degraded) - disulfide bond formation (strong bond) - attachment of prosthetic groups: sugars, functional groups, or lipids reversible modifications - phosphorylation - acetylation - methylation

Lysogenic cycle

phage genome integrates into bacterial chromosome, forming 'prophage' (integrated phage DNA) - prophage is not harmful to infected bacteria (temperate phage) - when bacteria (lysogen) is in a stressed condition, prophage escapes and follows lytic cycle

Lytic cycle

phage particles are multiplied from infected bacterial cell - bacterial cells undergo lysis

Promoter region (lacP)

polymerase binding site

Repressible regulation

presence of a particular molecule inhibits gene expression (repressor)

Repressor gene (lacI)

produces repressor (trans-acting factor) - no a part of the operon

Trans-acting elements

proteins that bind to the cis-acting sequences to control gene expression

trpR

regulatory gene of trp operon - codes for trp repressor that is active when the effector is bound and attaches to DNA

Anabolic genes in an operon are usually regulation in an _____ manner.

repressible

Operator region (lacO)

repressor binding site (cis-acting element)

No lactose present

repressor binds to operator region and no gene expression

Mutation on operator region (Oc)

repressor cannot bind on operator region - genes are always turned on

Mutation on repressor gene/protein (I-)

repressor cannot bind to the operator region -genes are always turned on

Negative regulation

repressor protein prevents transcription

Translational regulation

repressors and antisense RNA

Lactose

serves as an inducer of the genes expression

Mutation on repressor protein (Is)

superresprsessor - repressor protein lacks lactose binding site -operon is permanently repressed regardless of lactose present

Facultative gene

transcribed only when needed, constitutive genes are expressed continuously - the way majority of genes are regulated example: metabolism-related enzymes, responses to environmental stress, cell division related proteins 'enzyme adaptation' - the expression of a gene depends on the presence of a particular particle example: b-galactosidase production is dependent on the presence of lactose in media

Lysogenic cycle determinants

when cII-cIII complex accumulates to sufficient levels lysogenic cycle is favored - cII protein activates genes that encode λ repressor (cI) and integrase (int) - these two proteins promote lysogenic life cycle - integrase and λ repressor inhibit lytic cycle - starvation conditions favor lysogenic cycle

Lytic cycle deterinants

when cro protein level is high lytic cycle is favored - cro protein inhibits transcription from PRM, PrR, and PL related genes (inhibits λ repressor production) - cro protein also allows for the transcription of the O, P, and Q genes (involved in replication of λ DNA - when activity of cro protein exceeds that of CII protein, lytic cycle prevails

Jacob and Monod model (1961) on adaptive gene expression

worked with lactose metabolism in E. coli 1) Exposure to lactose causes a 1,000 to 10,000-fold increase in lactose metabolic enzymes 2) This is due to an increase in enzyme synthesis 3) Removal of lactose dramatically terminated enzyme production 4) Mutations indicated that separate genes are responsible for each enzyme

A riboswitch only affects translation of an operon. ⊚true⊚false

⊚false