Mmbio 240

catabolite repression

*due to EFFECT of GLUCOSE a second way that the lacO can be transcriptionally regulated 1. influenced by presence of glucose, a CATABOLITE: substance broken down inside cell 2. glucose leads to REPRESSION of lacO 3. when exposed to glucose and lactose, E.Coli 1st uses glucose and catabolite repression prevents use of lactose

O2 and O3 operator sites

*initially called pseudo-operators because substantial repression occurred in absence of wither one of them *BUT if O2 and O3 are missing, repression is dramatically REDUCED even when O1 is present *when O1 is missing, even w/ O2 or O3 around repression is nearly abolished!

cis-acting element

*mediated by DNA sequences BOUND BY REGULATORY PROTEIN *mutation in cis-acting element not affected by cis-acting element with intro of 2nd gene w/ normal function DNA segment that must be adjacent to the gene it regulates and has a cis-effect on gene expression ex of element: lacO site

trans-acting factor

*mediated by genes that ENCODE REGULATORY PROTEINS *mutation in trans acting factor can be complemented by intro of 2nd gene w/ normal function regulatory protein with TRANS EFFECT: genetic regulation that can occur even though 2 DNA segments are not physically adjacent ex of factor: lac repressor ex of effect: lac repressor on lacO

attenuations's stem-loop formation

1-2, 2-3, or 3-4 (but if 2-3, cannot use 3 for 3-4)

2 key features in attenuation

1. 2 trp codons found in mRNA that encodes trp leader peptide: codons provide a way to see if there is enough trp to synthesize proteins 2. mRNA can form stem-loop

inhibiting transcription through 2 ways

1. COREPRESSOR: small mlc that binds to a REPRESSOR and causes it to BIND to DNA 2. INHIBITOR: binds to ACTIVATOR and PREVENTS it from binding to DNA *REPRESSIBLE GENES

3 possible stem-loop structures formed from trpL mRNA under different conditions of translation

1. NO TRANSLATION: TRANS and TRANSC NOT COUPLED: so most stable form of mRNA occurs when 1 H bonds to 2 and 3 H bonds to 4 which forms terminator stem loop and transc terminated just past trpL *translation and transcription both coupled below but levels of trp vary *high levels of trp allows ribosome to reach trpL stop codon because there is ENOUGH charged tRNA(trp)! 2. LOW TRP LEVELS: TRANS and TRANSC COUPLED: ribosome pauses @ trp codons in trpL because low amount of charged tRNA (trp) present; pause blocks 1 of mRNA so 2 can H bond to only 3 thus 3-4 cannot form; RNA polymerase can transcribe rest of operon 3. HIGH TRP LEVELS: TRANS and TRANSC COUPLED: transl of trpL gene progresses to stop codon where ribosome pauses thus blocking 2 from H bonding so 3-4 able to form; TERMINATES TRANSCRIPTION at U-rich attenuator

2 binding sites

1. allosteric site: where the effector mlc binds to 2. active site: part of protein that binds to actual DNA

lacI gene encodes a diffusible repressor protein experiment: RESULTS

1. as expected yellow amount same as in the presence or absence of lactose because a defective lacI gene induced operon; lactose not needed 2. as expected lacI gene codes for repressor protein that can diffuse throughout cell and bind to any lac operon 3. hypothesis that lacI^(-) mutation resulted in synthesis of internal inducer REJECTED...because merozygote would have still made an internal inducer and lacOs in merozygote would have been expressed in absence of lactose

lacI gene encodes a diffusible repressor protein experiment

1. cells lysed by sonication 2. lactose analog, beta-o-nitrophenylgalactoside (BONPG) was added 3. BONPG is colorless, but when cleaved by beta-galactosidase the product is yellow 4. amount of yellow is a measure of amount of beta galactosidase that is being expressed from lac operon

recall 2 functions of DNA loop

1. contains -35 and -10 regions which are recognized by SIGMA FACTOR of RNA POLYMERASE 2. contains binding site for cAMP-CAP complex: protein within loop that will facilitate lac repressor binding to O1 and O3

allolactose

1. converted into by BETA-GALACTOSIDASE from lactose 2. small effector mlc to regulate lac operon

Key experimental observations supporting enzyme adaptation

1. exposure of bacterial cells to lactose increased lactose-utlizing enzyme 1000-10,000 fold 2. increase in activity of enzyme due to increased synthesis of enzymes 3. removal of lactose from environment caused termination of synthesis of enzymes 4. mutations that prevented synthesis of particular protein involved in lactose utilization showed a separate gene encoded each protein

2 inducible systems

1. lac operon 2. ara opern *regulated by sugar molecules that activate transcription (INDUCERS)

lac operon aka operator site was first identified by mutation that prevented lac repressor binding:

1. lacO^(-) 2. lacC^(c) regardless constitutive expression of lac operon even in strains that make normal lac repressor protein

processes regulated at genetic level

1. metabolism 2. response to environmental stress 3. cell division

AraC can act as:

1. negative regulator of transcription 2. positive regulator of transcription *depending on whether or not arabinose is present

1. cAMP 2. adenyl cyclase 3. CAP

1. small effector mlc that acts as an inducer 2. enzyme that uses ATP to produce cAMP and is inhibited when bacterium exposed to glucose 3. activator protein mediates cAMP on lacO

1 repressible system

1. trp operon *regulated by tryptophan a CO-REPRESSOR that binds to repressor and turns operator off *also keep in mind: abundance of charged tRNA (Trp) in cytoplasm can turn off trp operon via ATTENUATION

genetic regulatory proteins that respond to small effector mlcs have 2 functional domains

1. where protein binds to DNA 2. binding site for effector mlc

3-4 stem-loop of attenuation

1. with U-rich sequence acts as INTRINSIC terminator aka p-INDEPENDENT terminator 2. causes RNA polymerase to pause and U-Rich dissociated from DNA: TERMINATES TRANSCRIPTION @ U-rich attenuator *so if 2-3 were to form there would be no transcription termination because 3-4 could not form

trpL gene

1st gene in trp operon that encode peptide with 14 amino acids called leader peptide

binding of translational repressor

2 ways: 1. bind to vicinity of Shrine-Dalgarno sequence and/or start codon and sterically BLOCK ribosome from initiating translation 2. repressor protein may bind outside ^ region but STABILIZE mRNA secondary structure that prevents initiation

attenuation

2nd mechanism of regulating trp operon that includes trpL gene region *can occur in BACTERIA because transc and transl are coupled (ribosomes attach to 5' of mRNA after synthesis via RNA polymerase) *TRANSCRIPTION actually begins, but terminated before entire mRNA is made

binding of cAMP-CAP complex to DNA causes:

90 degree bend in DNA structure

Repressible mechanism

ANABOLISM: synthesis of small molecules *co-repressor or inhibitor *ex: tryptophan encoded by trp operon acts as co-repressor to bind to trp repressor protein when Trp levels high and turns OFF genes required for Trp biosynthesis when enough of the amino acid is made -->repression provides bacteria with a way to prevent overproduction of the product

AraC in absence of arabinose

AraC acts as REPRESSOR 1. AraC dimer binds to araO(1): INHIBITS TRANSCRIPTION of araC gene keeping AraC protein levels lows 2. AraC monomers bound to araI and araO(2): REPRESS ara OPERON ->araI and araO(2) can bind and form a loop in DNA thus PREVENTING RNA polymerase from binding to DNA and transcribing ara operon by P (BAD) ***ARA OPERON TURNED OFF

AraC in presence of arabinose

AraC as ACTIVATOR when arabinose bound to AraC protein 1. interaction between AraC at araI and araO(2) is broken opening up loop 2. another AraC protein binds to araI operator site and ACTIVATES TRANSCRIPTION by directly interacting with RNA polymerase *activation can occur in conjuction w/ activation of ara operon by CAP and cAMP if glucose levels are low ***ara OPERON ACTIVATED=bacteria can METABOLIZE ARABINOSE

Inducible mechanism

CATABOLISM: or breakdown of a substance *substance to be broken down acts as inducer *ex: allolactose as inducer for lac operon *ex: arabinose as inducer for ara operon *only expresses genes when needed to catabolize these sugars

presence of lactose and glucose

DIMINISHES TRANSCRIPTION 1. cAMP decreased so that its released from CAP and prevents CAP from binding to CAP site but diminishes because... 2. lactose causes lac repressor to be inactive

inducible system

OFF unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule is said to "induce expression". *Mlc turns on

repressible system

ON except in the presence of some molecule (called a corepressor) that suppresses gene expression. The molecule is said to "repress expression". *Mlc turns off

presence of only lactose

PROMOTES TRANSCRIPTION 1. cAMP high 2. lac repressor not bound to lacO

attenuation's termination of transcription:

RNA polymerase has not transcribed trp A,B,C,D,E and will NOT ENCODE PROTEINS for TRP BIOSYNTHESIS: *ATTENUATION INHIBITS FURTHER PRODUCTION OF TRP

polycistronic RNA

RNA that contains the sequence for 2 or MORE GENES that the OPERON encodes for

presence of only glucose

TRANSCRIPTION INHIBITED/VERY LOW 1. cAMP decreased so that its released from CAP and prevents CAP from binding to CAP site 2. glucose allows lac repressor to be bound to lacO *called CATABOLITE REPRESSION

operon

a group of 2 or MORE GENES under transcriptional control of a SINGLE PROMOTER *common in BACTERIA

Experiment showed:

a loss-of function mutation in a gene encoding a repressor protein has same effect as mutation in operator site that cannot bind a repressor protein *in both cases lacO genes are constitutively expressed

catabolite activator protein (CAP)

activator protein composed of 2 subunits each of which binds one mlc of cAMP and mediates cAMP effect on lacO

When lac repressor is active:

aka NOT BOUND TO ALLOLACTOSE: cAMP-CAP complex facilitates binding of lac repressor to O1 and O3 sites

merozygote

aka partial diplod is a strain of bacteria containing F factor genes *2 lacI genes in merozygote may be DIFFERENT ALLELES 1. lacI on chromosome may be lacI^(-) allele causing constitutive expression 2. lacI gene on F' factor may be normal

bacterial advantage of operon

allows bacterium to coordinately regulate a group of 2 or MORE genes that are involved with a common functional goal; expression of genes occurs as a single unit

how do genetic regulatory proteins recognize specific DNA sequences?

amino acid side chains directly interact with major groove of DNA helix *lac repressor tetramer(4 identical subunits) *each dimer w/ in tetramer recognizes operator site (2 sites such as O1 and O3)

only lactose present cAMP levels...

are HIGH 1. cAMP binds to CAP 2. CAP binds to CAP site and RNA polymerase can begin transcription

concentration of allolactose dictates ability to bind to the repressor:

as bacterial cell metabolizes sugars it lowers the concentration of allolactose below affinity for the repressor so lac repressor unlikely to be bound to allolactose *allolactose released, lac repressor can bind to operator which shuts down lac operon (due to depletion of lactose in environment)

cAMP is reduced when

bacterium is exposed to glucose because glucose stimulates signal pathway that inhibits ADENYLYL CYCLASE which is the enzyme needed for cAMP synthesis

When lac repressor is inactive:

bending of DNA appears to allow RNA polymerase to initiate transcription slightly downstream from the bend

absence of both lactose and glucose...

cAMP levels are HIGH *HOWEVER binding of lac repressor (due to glucose) INHIBITS transcription even though CAP is bound to DNA **transcription RATE LOW

polycistronic mRNA

carries several open reading frames (ORFs), each of which is translated into a polypeptide. These polypeptides usually have a related function (they often are the subunits composing a final complex protein) and their coding sequence is grouped and regulated together in a regulatory region, containing a promoter and an operator. Most of the mRNA found in bacteria and archea is polycistronic

F factor genes

carry genes originally found w/ in bacterial chromosome such as lacI gene and portion of lac operon *can be transferred from 1 cell to another by bacterial conjugation

1. metabolism

certain enzymes are needed for when bacterium is exposed to sugars in the environment and needs to metabolize it

2. response to environmental stress

certain proteins help bacterium to survive stresses such as osmotic shock or heat shock

when allolactose binds to repressor

conformational change occurs in lac repressor protein and PREVENTS IT from BINDING to operator site *operon has been INDUCED and allolactose's action as a small effector mlc is ALLOSTERIC REGULATION

lacI^(-) mutant

constitutive expression of the lac operon even in the absence of lactose

ara operon

contains 3 structural genes: 1. araB 2. araA 3. araD that encode a polycistronic mRNA for the 3 enzymes involved in arabinose metabolism *3 enzymes METABOLIZE arabinose into D-xululose-5-phosphate

ara operon like lac operon in that

contains a single promoter: P (BAD) *operon also contains a CAP site for binding of catabolite activator protein

lac operon

contains: a. CAP site b. promoter (lacP) c. operator (lacO) d. 3 structural genes: lacZ, lacY, lacA e. terminator

ability of lac repressor to bind to operator site

depends on if ALLOLACTOSE is BOUND TO IT *repressor's 4 subunits each have a single binding site for allolactose that acts as the INDUCER

lac repressor and CAP

determines whether lacO is expressed in presence or absence of lactose/and or galactose

small effector mlcs

do NOT BIND DIRECTLY to DNA to alter transcription, but BIND TO ACTIVATOR or REPRESSOR creating a CONFORMATIONAL change

lacA gene

encodes GALACTOSIDE TRANSACETYLASE: an enzyme that 1. covalently modifies lactose and lactose analogs 2. which through acetylation of non-metabolizable lactose analogs, may prevent toxic buildup within bacterial cytoplasm

lacY gene

encodes LACTOSE PERMEASE: membrane protein required for 1. active transport of lactose into cytoplasm of bacterium

lacZ gene

encodes enzyme BETA-GALACTOSIDASE: an enzyme that 1. cleaves lactose into galactose and glucose 2. converts small % of lactose into allolactose (structurally similar sugar)

trp operon

encodes enzymes needed for biosynthesis of amino acid tryptophan *genes trp: A,B,C,D,D encode enzymes SO UNLIKE lac operon that stops synthesis??

trpR gene

encodes trp repressor protein *when tryptophan levels are very low, trp repressor CANNOT bind to operator site *RNA polymerase transcribes trp operon *cell EXPRESSES genes required for synthesis of tryptophan VS *when levels are high, tryptophan acts as CO-REPRESSOR that binds to trp repressor protein *causes CONFORMATIONAL CHANGE in trp repressor ALLOWING it to BIND to trp operator *INHIBITS RNA polymerase to transcribe operon and operon TURNED OFF

O2

farther downstream in lacZ coding sequence

AraC

gene in the ara operon adjacent to ara operon and encodes regulatory protein called AraC protein that can bind to 1. araI 2. araO(1) 3. araO(2) * also has its own promoter P(c)

lac repressor

homtetramer: protein composed of 4 identical subunits *appx 10 homtetramer proteins per cell; only small amount of lac repressor protein needed to repress lac operon

regulatory protein names

how they affect transcription when bound to DNA (REPRESSOR or ACTIVATOR)

small effector mlc names

how they affect transcription when they are present in the cell at a SUFFICIENT CONCENTRATION to exert their effect

most common way for bacteria to regulate gene expression?

influencing RATE at which TRANSCRIPTION IS INITIATED *regulatory proteins can bind to DNA and affect the transcription of 1 or more nearby genes: 1. repressor: binds to DNA and inhibits TRANSCRIPTION (NEGATIVE CONTROL) 2. activator: increases rate of TRANSCRIPTION (POSITIVE CONTROL)

lacI gene

involved in transcription regulation but NOT PART OF lac operon *expressed at low levels *has its OWN promoter (i promoter) *encodes the LAC REPRESSOR: protein important for regulation of lac operon

when lactose present, lac repressor...

is NOT bound to lacO site so transcription can proceed @ HIGH RATE

If lacI (S) mutation: lac repressor is unable to bind to allolactose

lac operon cannot be induced even in the presence of lactose *super-repressor mutations typically located in domain that binds allolactose *usually lac repressor cannot bind to allolactose and therefore remains bound to lac operator site and induction CANNOT OCCUR

lacI (-): If lac repressor is unable to bind to DNA

lac operon cannot be repressed ...whereas...

maximal repression when

lac repressor binds to: A. O1 and O2 or B. O1 and O3

lac operon+lac repressor protein

lac repressor protein binds to sequence of nucleotides found within lac operator site *PREVENTS RNA POLYMERASE from TRANSCRIBING lacZ, lacY, and lacA genes *reversible process *in absence of allolactose, lac repressor is bound to the operator site most of the time

DNA forms a loop to promote binding of lac repressor to 2 operator sites:

loop forms because operator sites are distanced from each other *loop being operator sites closer together and facilitates binding of repressor protein *loop also inhibits RNA polymerase from sliding past O1 site to transcribe operon

lacO^(c)

mutations were localized in lac operator site known as O1

absence of lactose

no inducer available to bind to lac repressor *therefore lac repressor binds to operator site and INHIBITS TRANSCRIPTION *actually small amounts of BETA-GALACTOSIDASE, LACTOSE PERMEASE, and TRASACETYLASE are made

CAP site

recognized by activator protein called CATABOLITE ACTIVATOR PROTEIN (CAP) *short DNA segments that functions in gene regulation

translational regulatory protein

recognizes sequences within mRNA *mostly INHIBIT TRANSLATION and are known as TRANSLATIONAL REPRESSORS recall:(similar to transc. factors recognizing DNA sequences)

attenuator sequence

segment of DNA that helps TRANSCRIPTION TERMINATION

operator site

sequence of bases that provides binding site for REPRESSOR PROTEIN *short DNA segments that functions in gene regulation

diauxic growth

sequential use of 2 sugars by a bacterium *glucose preferentially metabolized *2nd sugar metabolized after glucose is depleted from environment

promoter

signals the beginning of transcription by coming beside an operon

O1

slightly downstream (towards 3') from promoter

O3

slightly upstream from promoter

bacteria exposed to lactose

small amounts of lactose transported INTO CYTOPLASM via 1. lactose permease 2. beta-galactoisidase converts some of it to allolactose *levels of allolactose rises and can eventually bind to lac repressor: 1. promotes conformational change that prevents repressor from binding to lac operator so that lacZ, lacY, and lacA can be transcribed 2. translation on encoded polypeptides produces proteins needed for lactose uptake and metabolism

cyclic AMP (cAMP)

small effector mlc produced from ATP by enzyme ADENYLYL CYCLASE *glucose is NOT ITSELF small effector mlc that binds directly to a genetic regulatory protein

inducer

small effector mlc that causes transcription to INCREASE by: 1. bind to REPRESSOR and PREVENT it from binding to DNA (so make it FALL OFF) 2. bind to ACTIVATOR and cause it to BIND to DNA *INDUCIBLE GENES

3. cell division

some proteins necessary only when cell is ready to divide

terminator

specifies end of transcription *2 or MORE genes found between promoter and terminator

trp mutant strains

still could INHIBIT expression of trp operon in presence of tryptophan *mutation in regions where trpL gene MISSING *mutations led to higher levels of other genes in trp operon

arabinose

sugar ARABINOSE consistuent of plant cell walls

lacI gene's rare mutations reveal

that since it alters the regulation of the lac operon the lac repressor is composed of a protein domain that binds to 1. DNA 2. another domain that contains allolactose-biding site

posttranslational regulation

the functional control of proteins that are already present in the cell rather than regulation of transcription or translational; it can: 1. activate protein 2. inhibit protein *relatively FAST: advantage

enzyme adaptation phenomenon

the observation that a particular enzyme appears within a living cell ONLY after the cell has been EXPOSED to a particular substance; when a bacterium is NOT EXPOSED to particular substance, it does NOT MAKE the enzymes needed to metabolize that substance

constitutive genes

unregulated genes *often they encode proteins continuously needed for survival of the bacterium