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FRNSC 420 - EXAM 4-Questions with Correct Answers/ Verified/ 100% Pass
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FRNSC 420 - EXAM 4-Questions with Correct Answers/ Verified/ 100% Pass
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FRNSC 420 - EXAM 4-Questions with Correct Answers/ Verified/ 100% Passpositive allosteric regulation - ✔️✔️binding of a regulatory molecule to an enzyme induces a
conformation change, increasing the enzyme's affinity for a substrate
- proteins are activators = enhance RNAP binding and/or induce conformational change, can change
binding constant (KB) or rate constant (kf), lowers activation energies
negative allosteric regulation - ✔️✔️bound regulatory molecule must release to induce the
conformation change necessary for substrate to bind an enzyme
- proteins are repressors = block RNAP binding or escape
allosteric regulation of Cdk - ✔️✔️replication initiation in eukaryotes
- Cdk is inactive when not bound to cyclin, with helices blocking its active site; when Cdk is inactive,
helicases are readily loaded onto DNA
- cyclin binds to Cdk to induce a conformational change that exposes its active site
- active site is phosphorylated by kinase CAK; when activated, Cdk will phosphorylate Cdc6/Cdt1 in
the eukaryotic PIC
- phosphorylation activates loaded helicases and prevents others from loading, allowing for the
unwinding of DNA and subsequent initiation of replication
effects of binding constants on promoters - ✔️✔️promoter/RNAP interaction strength can be
determined by the amount of the holoenzyme needed to occupy ½ of the promoter
- KB = [RP]/[R][P]
- equilibrium in bound & unbound RNAP is [RP]/[R][P] = 1
- from that, you get KB = 1/[R], shows KB when ½ of the promoter is occupied
- when KB is large, less holoenzyme is needed to bind ½ of the promoter = stronger RNAP/promoter
interaction
- when KB is smaller, more holoenzyme needed = weaker
- also dependent on rate constant k, where larger k = quicker conversion
allosteric regulation of glnA - ✔️✔️RNAP is pre-bound to glnA (glutamine synthetase gene, involved
nitrogen metabolism) in the closed complex
- RNAP has a σ⁵⁴ factor = unable to unwind DNA, must be activated
- activator is NtrC, binds 4 sites at ~150 bps upstream from promoter
,- IHF contains pseudo-loops which bind DNA and bend it 180⁰, facilitating interaction between NtrC
and the σ⁵⁴ factor
- NtrC utilizes ATPase activity; energy from ATP hydrolysis induces a conformational change in RNAP
to 'open' it
- process is regulated by environmental conditions in the cell; NtrC can only bind to DNA in low
nitrogen levels
- NtrC is phosphorylated by kinase NtrB, which induces a conformational change in NtrC to expose its
DNA-binding domain
- only then it can bind DNA and activate transcription
promoter spacing in merT - ✔️✔️protein MerR controls the expression of the merT gene,
expression is activated in the presence of mercury (Hg)
- MerR binds sequence between -35 and -10 regions of the merT promoter opposite to RNAP
(contains σ⁷⁰) for simultaneous binding
- spacing between the -35 and -10 elements is ~19 bps, much larger than the usual 15-17 bps;
elements are unaligned on DNA, facing opposite sides of the helix
- without Hg, MerR binds to lock this conformation in position; structural distortion prevents RNAP
from transcribing the gene
- when bound to Hg, MerR undergoes a conformational change that allows for DNA to twist at the
center of the promoter
- new configuration allows for the -35 and -10 elements to align with one another
catabolite repression - ✔️✔️in E. coli, presence of glucose causes the down regulation of operons
whose products participate in the metabolism of other carbon sources
- glucose is preferred carbon source in E. coli
- when lactose is absent, the lac repressor will remain bound to the lac operator, only dissociating
when lactose is present = basal transcription
- through induction (activation), transcription of the lac operon is further activated in the presence
of lactose when glucose is absent entirely
operons - ✔️✔️cluster of coordinated, regulated genes; contain:
- structural genes that encode enzymes
- regulatory genes that encode repressors or activators
- regulatory sites (promoters, operators, binding sites)
, lac gene expression products - ✔️✔️expressed at high levels ONLY when lactose is present and
glucose is not
- lacY: encodes lactose permease, inserted into the membrane to transport lactose into the cell from
the growth/surrounding medium
- lacA: encodes thiogalactoside transacetylase, removes toxic thiogalactosides (similar structure to
lactose) transported into the cell by lactose permease.
- lacZ: harbors the lac promoter on its 5' end and encodes β-galactosidase, cleaves lactose into
allolactose/hydrolyzes lactose to galactose and glucose
CAP in lac operon - ✔️✔️activates or increases the level of expression of the lac operon through a
glucose signal (absence of glucose)
- activated through the binding of cAMP
- CAP-cAMP complex binds the CAP site upstream of the promoter; an "activating region" physically
interacts with RNAP through cooperative binding w/ αCTD
- cAMP is down regulated by the presence of glucose; in these conditions, [cAMP] is low to prevent
activation of CAP
- only when glucose is absent will [cAMP] increase, allowing for activation of CAP
- lac operon is only fully activated in the absence of glucose AND the presence of lactose; if lactose is
absent, lac repressor remains bound = cannot be induced by allolactose or another artificial inducer
types of CAP protein - ✔️✔️- class I: recruitment, increases RNAP binding (KB), seen in lac operon
- class II: escape, increases rate of open complex formation (kf)
lac repressor - ✔️✔️binds the lac operator, a ~21 bp sequence containing a region of two-fold
symmetry (half-sites)
- lac repressor binds both half-sites as a homodimer containing helix-turn-helix regions; the
recognition helix binds major groove of DNA while the other α-helix interacts w/ the phosphate
backbone
- may also bind as tetramer to interact with two lac operators at once
- the operator overlaps the lac promoter; when the repressor is bound, it physically blocks RNAP
activity at the promoter
activated expression of lac operon - ✔️✔️lac operon is only actively expressed in the presence of
lactose AND the absence of glucose
- presence of lactose allows for the conversion of lactose to allolactose, acts as an inducer for the lac
repressor
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