Molecular Genetics Chapter 14

feedback inhibition - Metabolic Pathway

(1) Final product of the pathway inhibits activity of one or more enzymes in the pathway (2) Enzyme with regulatory- Allosteric Enzyme (site that contains two different binding site) (3) Binding of final product to regulatory site changes conformation of enzyme (4) Enzyme cannot bind to substrate

Translation and Post-translational regulation

(1) Majority of gene regulation in bacteria is at transcriptional level using least amount of energy (2) Posttranslational regulation is also possible inducer and inhibitors

covalent modification of enzyme

(1) Post translational covalent modification (2) Permanent modification may include, (a) Proteolytic processing digestion (b) Disulfide bond formation covalent modification (c) Attachment of: sugars, functional groups, or lipids (3) Reversible modifications: phosphorylation, acetylation, and methylation

repressors

(1) Repressor recognition sequences within mRNA (2) Repressors may bind to Shine-Dalgarno sequence ribosome binding site (3) Repressor binding may cause structural change on mRNA

Isolation of repressor

(a) By Walter Gilbert and Benno Muller-Hill (1966) (b) About 10 copies of lac repressor molecules in a cell (c) Developed Iq (regulator quantity) mutant -produces 10 times more repressor (d) Candidates were RNA and protein

possible stem-loop structures of trpL mRNA - tryptophan present

- Charged trp-tRNA present - Ribosome moves until it meets termination codon - Stem-loop formation of attenuator region - Transcription to trp operon is halted (no tryptophan production)

possible stem-loop structures of trpL mRNA - no translation on mRNA from trpL gene

- Stable stem-loop formation of attenuator region - No transcription and translation of trp operon

3 sites

- identified by mutation studies - repressor must bind to 2 out of 3 operator sites to repress transcription - O1 should be functional to repress O1 w/O2 or O1 w/O3 never O2 w/O3 - repressors from a tetramer and DNA loop

Types of riboswitches

- transcription - translation - RNA stability - Splicing

Catabolite-activating protein (CAP) is involved

1 - CAP facilitates RNA polymerase binding on promoter - stimulates gene expression 2 - 'Cooperative binding' -enhances each other's binding character 3 -CAP is also called 'cyclic AMP receptor protein' CAP binds to cyclic AMP or wont bind to DNA

Mutation on operator region (OC) (operator)

1 - Repressor cannot bind on operator region 2 - Genes are always turned on

Mutation on repressor gene/protein (I-)

1 - Repressor cannot bind to operator region 2 - Genes are always turned on

Experiments by Jacob, Monod and Pardee tested for one of two following possibilities

1- lacI gene encodes a diffusible repressor protein 2 - lacI- mutation synthesizes an internal inducer

operon is flanked by

1- promoter = signals the beginning of transcription 2 - terminator = specifies the end of transcription

mutations on repressor gene or operator region

A) Mutation on repressor gene/protein (I-) B)Mutation on repressor protein (Is): superrepressor (superrepressor) C) Mutation on operator region (O-) D) Mutation on operator region (OC) (operator)

lac operon contains?

CAP site lac promoter (lacP) operator site (lacO) three protein-encoding genes lacZ, lacY and lacA terminator

glucose & lactose absent

OFF CAP and Repressor bound

positive regulation

activator protein promotes transcription - binding of activator promotes transcription - regulation by an activator

each of reproessor's protein four subunits has a single binding site for?

allolactose, the inducer

small effector molecule such as allolactose is called

allosteric regulation

post-translational regulation

regulation of availability of the protein made

anabolic genes are regulated in an operon how?

repressible manner

negative regulation

repressor protein prevents transcription - transcriptional regulation by a repressor

Translation regulation

repressors antisense RNA

corepressor

small effector molecule that decreases gene expression by binding to repressors

corepressor and inhibitor both act to reduce the rate of transcription

therefore they are termed repressible genes

lacA gene encodes?

transacetylase - removes by toxic by-products from lactose digestion

facultative gene

transcribed only when needed, constitutive genes are expressed continuously - majority of genes are regulated - EX: metabolism related enzymes, responses to environmental stress, cell division related proteins

CAP site: Catabolite Activator Protein binding site

(a) DNA sequence recognized by catabolite activator protein (b) CAP facilitates RNA polymerase binding on promoter (c) CAP binding to CAP site is cAMP dependent cyclic AMP is needed

Genetic proofs to support operon model

(a) Repressor gene produces a diffusible cellular product (proteins) (b) Operator region is involved in regulation, but no gene product is known (c) Operator region is adjacent to the structural genes to regulate transcription

trp operon model 1) No/Low tryptophan present

(a) Repressor is inactive -Cannot bind to operator region (b) Gene is expressed

trp operon model 2) Tryptophan present -Repression

(a) Tryptophan binds to repressor (Active form) -tryptophan is 'corepressor' (b) Active repressor binds to the operator region (c) Genes cannot be expressed

Osmoregulaiton

(a) ompF gene produces protein that forms porin on outermembrane of E. coli (b) Porin controls osmotic pressure of the cell - Porin plugs pores to keep solutes from going into the cell (c) In a hypotonic cell condition, solute should be introduced into the cell - Porin formation is inhibited by transcription of micF gene - RNA of micF gene is antisense to ompF mRNA - micF gene does not produce protein

Antisense RNA

- Complementary to mRNA - osmoregulation in E. coli

Catabolic repression/CAP inducible system

- Glucose is a catabolite of lactose digestion - Diauxic growth of E.coli

possible stem-loop structures of trpL mRNA - 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)

trp operon model 3) Tryptophan present -Attenuation - attenuator sequence

- Stem-loop formation of attenuator region causes premature termination of transcription - First two trp codons control ribosomal movement by availability of charged trp-tRNA

Mutation on repressor protein (Is): superrepressor (superrepressor)

1 - Repressor protein lacks lactose binding site 2 - Operon is permanently repressed regardless of lactose presence

Experimental system (Fig.14.7a, b)

1 - Used merozygote (partial zygote): Contains F' factor plasmid 2 - Result *(Table14.1) (lac mutation) 3 - Conclusions - Repressor is trans-acting factor - Operator region is cis-acting element

regulatory proteins are?

1 - activators 2 - repressors

organization of trp operon

1 - stuctural genes 2 - regulatory and repressor gene 3 - trp gene

Cycle of lac operon induction and repression

1 - when lactose becomes available a small amount is taken up and converted by beta-galactosidase - allolactose binds to the pressor causing it to fall off the operator site 2 - lac operon proteins are synthesized - this promotes the efficient uptake and metabolism of lactose 3 - the lactose is depleted - allolactose levels decrease and allolactose is released from the repressor allowing it to bind to the operator site

Mutation on operator region (O-)

1 -Mutation blocks passage of RNA polymerase 2 - Genes are always turned off

cis acting elements

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

transcription (riboswitches)

Formation of rho-independent transcription termination hairpins

RNA stability (riboswitches)

Formation of self-degradable ribozyme

dual control of lac operon

Glucose inhibits adenylyl cyclase activity( NOT ACTIVE) (glucose-cAMP) wont be able to produce cAMP - so CAP protein cannot bind to CAP binding site - RNA polymerase wont bind or get help from CAP = gene should (could) not be expressed

translation (riboswitches)

Inaccessible Shine-Dalgarno sequence (ribosome binding site)

three operator sites where lac repressor binds

O1 (most important for repressor to bind O2 O3

glucose present & lactose absent

OFF repressor bound

glucose & lactose present

OFF/(ON but low expression) CAP and Repressor off

glucose absent & lactose present

ON CAP bound

structural genes

a - Five continuous structural genes (TrpE, D, C, B, and A gene) b - Transcribes polycistronic mRNA c - Genes produce tryptophansythetase

CAP site =

a DNA sequence recognized yby an activator protein called catabolite activator protein (CAP)

operon

a group of two or more genes that are transcribed from a single promoter - unit of genetic material that functions in a coordinated manner by a single promoter

binding of repressor to operator site is

a reversible process

allolactose acts as?

a small effector molecule for regulating the lac opero

beta-galactosidase also converts?

a small percentage of lactose into allolactose

regulatory and repressor gene

a) Promoter region (P) b) Operator region (O) c) trpL gene Encodes leader peptide (14 amino acids) - mRNA determines transcription and translation of trp operon d) Attenuator region - Controls immature transcription of trp operon under the presence of tryptophan

why do operons occur in bacteria?

allows a bacterium to coordinately regulate a group of two or more genes that are involved with a common functional goal - expression of genes occur as a single unit

operon includes a polycistronic mRNA

an RNA that contains the sequences of two or more genes

lactose serves as?

an inducer of the genes' expression

lacZ gene encodes the enzyme?

beta-galactosidase - converts lactose to glucose and galactose

operator site lacO =

binding site for a repressor protein called lac repressor - cis-acting element

in absence of allolactose, lac repressor is?

bound to the operator site most of the time

Riboswitches

discovered in 2001 and 2002 - Binding of small molecule to a regulatory segment of mRNA causes conformational change and leads modification of mRNA function - Riboswitch of mRNA regulates its own translation activity and enzymatic activities

Effector Proteins

do not bind directly to DNA but react with repressors or activators - cause conformational change in activator or repressor

inhibitor

effectors that bind to an activator protein and prevents it from binding to the DNA

trp operon - repressible operon

encodes enzymes involved with biosynthesis of tryptophan - amino acid

adaptive (facultative) expression 'enzyme adaptation'

expression of a gene depends on presence of particular substance

inducible regulation

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

constitutive expression

gene is expressed continually regardless of the environment

constitutive gene

gene that is transcribed at a relatively constant level regardless of the cells environmental conditions (always expressed: control)

no lactose present

genes will be expressed - repressor binds to operator region & no gene expresion

lactose present

genes will be expressed (a) Lactose (allolactose) binds to repressor (b) Binding leads to the conformational change in repressor (c) Repressor cannot bind to operator region allowing transcription to occur (d) Then, the genes expressed

E. coli consumes what first?

glucose when glucose and lactose are available -When glucose present, lac operon doesn't express even though lactose is present

repressor gene lacI

has its own promoter called an i promoter and is constitutively expressed at fairly low levels

lac operon can be transcriptionally regulated how?

in more than one way

1 - activator

increase transcription causing - Positive Control - may be naturally active or inactive

lac operon regulation

inducible and negative control system

genes regulated by inducers are called?

inducible genes

catabolic genes are regulated in an operon how?

inducible manne

2 - repressor

inhibit transcription process causing - Negative Control - may be naturally active or inactive

first mechanism inducible and under negative control

involves lac repressor - which binds to sequence of nucleotides found within the lac operator site - once bound lac repressor prevents RNA polymerase form transcribing the lacZ, Y and A

if lac repressor is unable to bind to the DNA

lac operon can not be repressed

lacI gene encodes?

lac repressor, a protein that regulates the lac operon by binding to the operator site and repressing transcription

for maximum transcription of lac operon

lactose binds to repressor - CAP binds to CAP binding site +CAP binding is cAMP dependent +cAMP is needed for CAP to bind to CAP binding site +cAMP is produced from adenylyl cyclase

lacY gene encodes?

lactose permease, - facilitates entry of lactose into bacterial cell; co-transports H+

gene regulation

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

lacI- mutations that result in constitutive expression of lac operon -

means that it is expressed in both the presence and absence of lactose

trp gene

not a part of trp operon - produces repressor that binds to operator region

lacI^s mutations have the

opposite effect - the lac operon can be induced even in the presence of lactose

promoter region lacP =

polymerase binding site

repressible regulation

presence of a particular molecule inhibits gene expression (repressor)

inducer

small effector molecule that increase gene transcription by binding to activators - can bind to a repressor protein and prevent it form binding to the DNA - can bind to activator protein and cause it to bind to the DNA

in the case of lac repressor, the binding of allolactose alters?

the function of lac repressor by preventing it from binding to the DNA

transcription occurs when>

the repressor fails to bind to the operator region - gene is off by default

when four molecules of allolactose bind to the repressor a conformational change occurs that prevents lac repressor from binding to the operator site

under these conditions RNA polymerase is free to transcribe the operon - operon has been induced

ability of lac repressor to bind to the operator site depends on?

whether or not allolactose is bound to it

Jacob and Monod model on adaptive gene expression - 1961

worked with lactose metabolisms in E. coli 1) Exposure to lactose causes a 1,000 to 10,0000-fold increase in lactose metabolic enzymes 2) This was 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