molec 2 Ch. 20

which environmental conditions positively/negatively regulate the lac operon?

positive: lactose
negative: glucose

what do each lac operon genes (lacZ, lacY, lacA) code for?

lacZ: ?-galactosidase
lacY: galactoside permease
lacA: thiogalactoside transacetylase

which of the following is not part of the lac operon? what does it encode?
lacA, lacI, lacY, lacZ

lacI: encodes the lac repressor

T/F the lac operon codes for anabolic enzymes

false
catabolic enzymes
breaking down lactose

T/F the promoter and operator sequences of the lac operon slightly overlap

true

what 5 sequences make up the lac operon?

promoter, operator, lacZ, lacY and lacA

T/F lactose is the inducer of the lac operon

false
allolactose
binds to the lac repressor

what enzyme allows lactose to enter the cell?

lac permease

once lactose is in the cell, what are its two possible fates?

cleavage by ?-galactosidase into:
* galactose and glucose
* allolactose

when there is no lactose present, what does the lac repressor bind to?

binds to the operator (which overlaps the promoter) preventing RNA pol from binding

when lactose is present, what happens to the lac repressor?

allolactose binds to the repressor, inhibiting it from binding to the operator, allowing RNA pol to bind to the promoter (basal if glucose is also present)

how is the lac operon operator sequence composed?

21 bp sequence with two fold symmetry
three operators:
*major operator (O1): overlaps the . promoter
*Auxiliary operators (O2, O3): one is 400 bp downstream while the other is 90 bp upstream

how does the lac repressor bind to the lac operon operator sequences?

one dimer binds to the major operator (O1) and the other one binds to the a minor operator (O2 or O3) wha

what is the lac activator? what environmental condition causes this activator to be expressed?

CAP (which binds to cAMP, and CAP:cAMP binds DNA)
low glucose --> high cAMP which is derived from ATP

T/F cAMP is a global activator

true
can bind to more than 100 promoters
involved in the activation of other sugar metabolizing genes

which part of the RNA pol does the CAP:cAMP complex interact with?

sigma70 domain

what does CAP stand for? what is another name for CAP?

CAP = catabolite activator protein
CRP = cAMP receptor protein

T/F CAP is regulated by the lac operon

false
not regulated by lac operon, like lacI

where is the CAP-binding site relative to the lac operon promoter?

upstream

what does CAP-cAMP binding to the CAP-binding site do to help promote transcription?

stabilize the RNA pol:DNA interaction
no longer basal transcription, now being actively transcribed

what is commonly used experimentally as a lac operon inducer?

isopropyl ?-D-1-thiogalactopyranoside (IPTG)

how does IPTG induction of the lac operon allow researchers to study the regulation of the lac operon?

IPTG is cannot be metabolized and it can diffuse into the cell without permease and bind to the lac repressor, allowing the lac operon to be turned on
no need for allolactose (and therefore lactose) to be present

what allows for IPTG to not be hydrolyzed from the repressor like allolactose would?

IPTG contains a sulfur group

T/F the ara operon is regulated by CAP:cAMP similarly to the lac operon

true

what are the activators required for the activation of the ara operon?

CAP and AraC

what happens in the ara operon when arabinose is not present?

AraC binds as a homodimer to araI1 and araO2
this causes the DNA to form a loop, preventing RNA pol from binding

what happens in the ara operon when arabinose is present?

arabinose binds to AraC
Arac:arabinose dimer binds as a homodimer to araI1 and araI2
DNA is now linear
no basal transcription though because AraC is not enough for RNA pol to bind and initiate transcription
(requires CAP:cAMP)

what happens in the ara operon in the presence of arabinose and absence of glucose?

arabinose binds to AraC
Arac:arabinose dimer binds as a homodimer to araI1 and araI2 making the DNA linear
CAP:cAMP bind upstream of the araI site and RNA pol can bind and actively transcribe genes

T/F a major difference between the lac operon and ara operon is that in the lac operon there is occasional leakage allowing for basal transcription of the lac genes, but in the ara operon there is no basal transcription

true

T/F a repressor can hold the RNA pol at the promoter inhibiting transcription

true
prevents promoter escape

how does the Gal repressor (GalR) prevent the transcription of galactose metabolizing genes?

in the absence of galactose it binds to its DNA site, allosterically affects the RNA pol not allowing for the transition from the closed --> open complex

in the absence of glucose and present of galactose, where does CAP:cAMP bind relative to the gal site?

upstream
inhibits Gal repressor from binding so RNA pol can move from the closed --> open complex

T/F the control of the Gal operon is due to a physical change to the actual DNA and not the RNA pol

false
Gal operon due to RNA pol allosteric change
merT operon due to DNA change

what activates merR? does it interact with RNA pol?

activated by mercury
does not interact with RNA pol, binds directly do DNA

where does merR bind?

to a sequence between -10 and -35

why can't RNA pol bind to transcribe the merT operons?

there is 19 bp between the binding sites when is not optimal for sigma 70 to bind.
the distance of 19 bp also causes -35 and -10 to be on opposite sides of the DNA

how does the interaction of merR and Hg allow for RNA pol to bind and transcribe the merT operon?

twist the DNA so -35 and -10 are closer together and on the same side of the DNA

how many enzymes are in the trp operon? what are the enzymes used to make?

5 enzymes to make tryptophan

what is the precursor of tryptophan?

chorismate

T/F the trp operon codes for anabolic enzymes

true

T/F the trp operon is turned on in the presence of a substance

false
turned off in the presence of tryptophan

which of the following is NOT part of the trp operon: operator, promoter, leader region, CAP binding site, attenuator

CAP binding site

which of the following operons is regulated by attenuation:
merT, lac, gal, ara, trp

trp operon
repression alone only decreases transcription by 70x, attenuation by 10x gives a total repression of 700x

why can't attenuation occur in eukaryotes?

only occurs in cells without a nucleus because it depends on the coupled transcription/translation found in prokaryotes

what type of sequence controls the trp operon? where is this sequence located?

a leader sequence (trpL)
5' end of the mRNA

which of the following are structural genes in the trp operon:
trpP, trpO, trpL, attenuator, trpE, trpD, trpC, trpB, trpA

trpL, attenuator, trpE, trpD, trpC, trpB, trpA

what important sequences are located in the trpL, trpP and trpO genes?

trpL = leader sequence
trpP = promoter sequence
trpO = operator

what is an aporepressor?

a repressor with a piece missing

what occurs when there is no Trp present in the environment?

inactive aporepressor is present
no trp repressor
transcription of trp operon occurs

what occurs when there are low levels of Trp present in the environment?

Trp dos not bind to the aporepressor --> no trp repressor
inactive aporepressor is present
transcription of trp operons occurs

what occurs when there are high levels of Trp present in the environment?

Trp binds to the aporepressor --> trp repressor binds to the trp operator
no transcription of the trp operon

T/F Tryptophan is the repressor for the trp operon

false
corepressor

what is transcription attenuation?

regulation of bacterial operons by controlling termination of transcription at a site located before the first structural gene

where is the attenuator located in the trp operon?

within the leader sequence

which regions of the trp operon leader sequence form a secondary structure? what secondary structure do they form and what does it facilitate?

regions 3 and 4 form a stem-and-loop structure allowing for Rho-independent termination

what is located in region 1 of the trp operon leader sequence? what are the function of these genes?

Trp codons
if the cell has enough Trp, then tRNAs are loaded with Trp
when the ribosome gets to these codons, the tRNAs load Trp and when the ribosome gets to the stop codon at the end of region 1, it falls off
if there is not enough Trp in the cell then

in the trp operon leader sequence, what follows region 4?

poly(U) sequence

which regions form the terminator hairpin(s) in the trp operon?

1 and 2 (which includes AUG and stop codon
3 and 4 (includes attenuator sequence)

which regions form the anti-terminator hairpin(s) in the trp operon?

2 and 3
contains the two Trp operons where the ribosome stalls and does not allow 1-2 or 3-4 to form

does transcription or translation affect the pairing choice for the leader sequence hairpins?

translation

why does transcription/translation have to occur simultaneously for attenuation to work?

depends on the ribosome reaching the Trp codons in region 1 for the ribosome to get stuck or not
if the ribosome gets stuck on region 1 then 1-2 and 3-4 cannot come together to terminate transcription. regions 2-3 come together instead forming the anti-te

what occurs with the attenuation of the trp , when there is a high concentration of Trp present?

the ribosome translates the leader region until the stop codon between segments 1 and 2; segments 3 and 4 interact (terminator)
Rho independent termination
no trp operon transcription

what occurs with the attenuation of the trp , when there is a low concentration of Trp present?

the ribosome pauses at the trp codons in segment 1, allowing segment 2 to interact with 3 and form the hairpin (anti-terminator) --> trp operon transcription

what cellular response are the LexA repressor and RecA protein involved with?

SOS response
genes needed for repair (DNA damage)

what polymerases are also involved in the prokaryotic SOS response?

translesion synthesis (TLS) polymerases

what is the function of LexA? what causes LexA to dissociate?

inhibits the transcription of SOS genes by binding to promoters
autocleavage with the help of RecA causes the dissociation of LexA

what does RecA bind to? what does this cause?

single stranded (damaged) DNA
free LexA cleaves itself, and the lack of functional LexA allows SOS genes to be transcribed

where are transcriptional riboswitches located in bacteria?

5' UTR of mRNA

what regulatory elements act as direct sensors of small molecules, tRNA, temperature, pH or metals?

riboswitches

what two modules make up a riboswitch?

aptamer: where the ligand binds
expression platform: changes conformation of the mRNA causing a change in gene expression

how do riboswitches control transcription/translation?

transcription termination
sequestering of the SD sequence

how does a transcription riboswitch work?

a metabolite binds causing a terminator hairpin loop structure to form, inhibiting transcription

how does a translation riboswitch work?

metabolite binds causing the mRNA to dimerize, sequestering the SD sequence so it cannot be recognized by small subunit of ribosome, inhibiting translation

how are E. coli ribosomal protein operons regulated?

feedback translational control
r-proteins bind to RBS inhibiting small subunit from binding and making more un-needed r-proteins

what is L4? what does it do if there is an overabundance of r-protein present in a cell?

L4 = r-protein
binds to the polycistronic mRNA and inhibits the translation of r-proteins

where do r-proteins bind when there is free rRNA present in the cell? what about if there is no rRNA in the cell?

free rRNA: r-proteins binds to rRNA with high affinity
no rRNA: protein binds to second highest affinity location on mRNA

what is the most commonly chosen infection path a bacteriophage chooses? lytic or lysogenic?

lytic

are prophages formed in a lytic or lysogenic infection?

lysogenic

what is lysogenic induction?

when the phage switches from lysogenic to lytic stage

what type of bacteriophage infection kills the bacteria to release the phages progeny?

lytic infection

T/F in lysogenic bacteriophage infection, every time the bacteria replicates, the phage also replicates

true

when a bacteriophage moves from the lysogenic to lytic stage, what does the 5'-3' exonuclease do?

removes ssDNA

T/F the decision to move from lysogenic to lytic is reversible at any time

false
irreversible

what type of infection (lytic or lysogenic) occurs when only one lambda phage infects a cell? what about >2 phages?

1 phage: lytic
>2 phages: lysogenic

what type of infection (lytic or lysogenic) occurs if the lambda phage infected cell is slow growing? what if there is UV light damage?

slow: lysogenic
UV light: lytic

does lambda phage contain a RNA or DNA genome? is it encapsulated or unencapsulated?

DNA encapsulated phage

what are PL and PR promoters? when are they ON?

rightward and leftward lambda phage promoters
ON during lytic cycle
do not require the help from an activator

T/F PR and PL promoters require an activator to allow the lambda phage to undergo the lytic cycle

false
no activator help needed

what is PRM? when is it ON?

promoter for repressor maintenance
ON during the lysogenic cycle
Only transcribes the lambda repressor (cI) with the help of an activator

T/F the PRM promoter requires an activator to allow the lambda phage to undergo the lytic cycle

false
requires activator but for the lysogenic cycle

T/F PRM is a stronger promoter than PL and PR

false
weaker

what does the cI codon code for in the lambda phage genome? when is it transcribed?

lambda repressor
ON during lysogenic phase

what is the function of lambda repressor (cI)?

repressor of PR but an activator of PRM

what is located in the N and C-termini of the lambda repressor?

N-term: DBD (helix-turn-helix)
C-term: used to form homodimer that binds to DNA

which part of the lambda repressor interacts with RNA pol to activate transcription of cI?

N-term interacts with sigma region of RNA pol that binds to the -35 region

T/F the goal of the lambda repressor is to maintain the lytic state

false
maintain lysogenic state

what does the lambda repressor repress? what does it promote?

represses expression of cro
promotes its own expression (cI)

what does the CRO repressor repress? what does it activate?

it is a repressor only
controls the lambda repressor
does not act as an activator

T/F the CRO repressor only contains a DBD

true

T/F the CRO repressor binds to DNA has a homodimer and inhibits RNA pol from binding

true

what happens if PR is not inhibited by lambda repressor?

cro gene gets transcribed producing the CRO repressor

how many operons can lamdba repressor and Cro bind to?

one of six:
three on right hand (OR) control region: OR1, OR2, OR3
three on left hand (OL) control region: OL1, OL2, OL3

which operon does Cro have the highest affinity for?

OR3

which operon does lambda repressor have the highest affinity for?

OR1

OR1 and OR3 are located in which promoter regions?

OR1 is located in PR
OR3 is located in PRM

T/F lambda repressor forms a tetramer whose formation is mediated by the dimers N-terminus

false
C-terminus

T/F lambda repressor binds cooperatively to DNA

true

T/F lambda repressor binding to OR1 allows cooperative binding to other ORs by increasing affinity for OR2/OR3

true

T/F when RNA pol binds to PRM with the help of lambda repressor, PR and PL are ON which leads to the making of more lambda repressors

false
PR and PL are off
RNA pol binding to PRM allows for transcription of cI which codes for more lambda repressors

what does lambda repressor binding at OR1 cause? what about its binding to OR2?

OR1: inhibition of RNA pol binding to PR --> no Cro
OR2: activation of RNA pol binding to PRM --> activates cI to make more lambda repressors that can bind to OR1 and OR2

T/F higher concentration of lambda repressor turns off its own transcription

true

which operator site gets bound to when lambda repressor is present in high concentrations? what does this lead to?

OR3 is bound and if the other operators (Or1, OR2) are saturated then it leads to a halt in lambda repressor transcription

how does lambda repressor stabilize the lysogenic state?

by bringing together OR with OL

does the Cro dimer binding to OR3 control the lytic or lysogenic cycle?

lytic cycle

T/F Cro dimer binding to OR3, overlaps PR, suppressing PR and no lambda repressor is made

false
overlaps and suppresses PRM

T/F in lytic cycle control, Cro is bound to OR1 and OR2

false
no lambda repressor or Cro is bound to OR1 or OR2

T/F Cro binds cooperatively to DNA, increasing its binding affinity for OR2 by binding to OR1 for which it has an intrinsic high binding affinity

false
no cooperative binding by Cro

when RNA pol binds to PR and PL, what gets transcribed?

lytic genes transcribed
lytic cycle control

when there is DNA damage, what protein gets activated? what does this protein then cleave?

RecA cleaves LexA

what bacteriophage TF does LexA look like? what happens to this TF?

lambda repressor
gets cleaved by RecA
lambda repressor keeps the cell in lysogenic state, but when it is cleaved it gets released allowing for the transcription of PR and PL, synthesis of Cro, and then the phage switches to lytic growth

when switching from lysogenic to lytic, what interaction ensures that the switch is irreversible?

Cro binding to OR3, blocking further synthesis from PRM --> no more lambda repressor

the transcription of which proteins aid in the decision between lysogenic or lytic cycle upon infection of a new host cell? where are these proteins transcribed from?

cII transcribed from PR
cIII transcribed from PL

what is cII? where does it bind? what does it do?

transcriptional activator that binds to PRE (repressor establishment), a weak promoter
when the concentration of lambda repressor increases it binds to PRM and maintains its own transcription --> lysogenic cycle
stimulates the transcription of cI and Cro

what do alternative sigma factors regulate?

the ordered expression of genes

how is the expression of Spo1 phage sigma factors organized?

as a cascade
one sigma factor is required to transcribe the gene coding for the next sigma factor

what occurs in the first 5 minutes of the lytic cycle of Spo1 phage?

early genes of phage Spo1 are transcribed by the hjost RNA pol + host sigma factor

what occurs in the 5-10 minutes after infection of the host cell in the lytic cycle of Spo1 phage?

one of the early genes (gp28) codes for a sigma factor --> RNA pol + gp23 transcribe the middle genes

what occurs 10 minutes on, after the infection of a host cell in the lytic cycle of Spo1 phage?

middle genes gp33 and gp34 code for subunits of another sigma factor --> RNA pol + gp33:gp34 transcribe the late genes
phage takes over the cell

why does Spo1 phage use the host RNA pol at first?

because it doesn't carry its own sigma factor

T/F the goal of Spo1 phage is to use to host cell to transcribe only its own genes and none of the hosts

true

in early sporulation, what factor is being made?

a different sigma factor than before

in early sporulation, which sigma factor stays behind, and which one goes into the spore?

sigma E stays, sigma F goes into the spore

ultimately, what sigma factor remains in the spore and what remains in the mother cell?

sigma G stays in the spore
sigma K stays in mother cell
ultimately only spore is left

T/F once the decision to undergo sporulation has been made, it can be reversed only before it enters the late sporulation stage

false
cannot stop