BIOL 300 - Chapter 24

what are the factors of eukaryotic gene regulation?

has multiple levels of regulation, long term but slow (theres regulation for quick responses to variety of signals tho), and the gene default state is inhibited

what are the factors of prokaryotic gene regulation?

limited need for regulation, but what regulation occurs focuses on transcription regulation, since mRNA are unstable and are immediately available. the gene default state is activated

how is regulation different than transcription factors?

TFs control the core promoter and whether RNAP will get to the promter. Regulation that occurs upstream influences how the processes downstream occur

what is a cis acting element?

regulates the same gene they belong to on the DNA strand (promoter, terminator)

what is a trans acting factor?

a gene that’s transcribed into a regulator protein that influences another DNA gene sequence (sigma/transcription factors)

what is an operon?

transcription unit that has several genes underneath the control of a shared promoter/terminator, so genes that work together can be regulated by the same promoter. each gene has a separate start and stop codon, but it allows genes of related functions to be regulated together

what does an operon produce?

polycistronic mRNA

what makes up the lac operon?

promoter, operator, LacZ, LacY, LacA

what is the role of a repressor?

prevents RNAP from going down the DNA, by binding to the operator in a negatively controlled site

what is the role of an activator?

helps start transcription by binding to the activator binding site (NOT PROMOTER) in negatively controlled site

how do small molecules inactivate/activate trans-factors?

binding and inducing conformational changes in the trans-factors upstream

how do trans-factors inactivate/activate gene expression?

by binding to the cis elements associated with genes downstream

what is the pathway of bacterial gene regulation (broad view)?

signals activate small molecules, which can activate/inactivate trans factor (repressor or activator), which will then bind/not bind to its cis element, and affect target gene expression levels

what is the process of induction in negatively controlled genes?

cis element will encode a repressor, which will always be on the DNA. the repressor will be ON, so an inducer (allolactose) will bind and remove it from the DNA to allow the gene to be transcribed

what is the process of repression in negatively controlled genes?

cis element will encode an inactive repressor, so it will be OFF. A corepressor will ned to bind to the repressor to activate it and stop transcription.

what is the process of induction in positively controlled genes?

a cis element will make an activator, which will be OFF. It will need an inducer to bind to allow it to bind to RNAP, in order to enhance transcription

what is the process of repression in positively controlled genes?

a cis element will make an activator, which will be bound to DNA and be ON. A corepressor can bind to it and inactivate it, repressing transcription

what makes up the negative control?

operator, and repressor

what makes up the positive control?

activating binding sequence, and activator

what is the transcription control for the lac operon?

has two initiation controls, both of which must be turned on

what is the transcription control for the Trp operon?

has two controls, one for initiation and one for termination, and has two levels of expression in parallel regulation

what do the lac operon products allow?

allows bacterial cells to take up and metabolize dissacharide sugars like lactose

what does lacZ encode in the lac operon?

gene that breaks the beta-galactosidase bond in the dissacharides, to allow it to break down

what does lacY encode in the lac operon?

permease that allows for lactose to be transported into the cell

how is the lac operon under dual control of initiation?

a repressor needs to be removed by inactivating it with an inducer, and the activator needs to be activatd with an inducer

what happens in the lac operon if glucose is present?

enzymes involved in using beta galactoside aren’t needed, and the lac operon shouldn’t be activated

what conditions will allow the lac operon to be induced?

in the absence of glucose and the presence of lactose

is the lac operon still expressed without beta galactoside?

yes, but in very low amounts

what are the mRNA and protein levels of the operon after beta galactosidase are present and then removed?

mRNA levels will rise and drop fast in time with the presence of beta galactoside, but protein levels will lag after rising before dropping again

how does the lac operon have negative inducing?

gene is normally turned off by the repressor binding to the operator, and inducer binding inactivates the repressor

what three domains does the repressor have?

DNA binding domain, core subdomain, and tetramerizatoin domain

how does an inducer prevent the repressor from acting?

inducer will bind to the core domain, change its protein conformation and prevent it from binding to the operator

what is the process of allosteric control?

preventing the inducer from binding to the repressor

what interactions can mutations in the operator/repressor prevent?

operator and repressor, inducer and repressor, or repressor and repressor

what types of operon expression are present if a mutation in the operator occurs?

constitutive expression (always on)

what types of operon expression are present if a mutation in repressor DNA binding domain occurs?

constitutive expression (always on)

what types of operon expression are present if a mutation in repressor inducer binding domain occurs?

constitutive repression (always off)

how does the lac operon have positive inducing?

the operon won’t be expressed even if the repressor is absent, because the promoter is weak and doesn’t bind to polymerases well - that’s why an activator is required for further stabilization           

what is the lac operon activator?

CRP (cAMP receptor protein)

what is the lac operon activator’s inducer?

cAMP

what happens to cAMP in the presence of glucose?

adenylate cyclase is prevented from producing cAMP

what is the relationship between glucose and cAMP?

as glucose goes up, cAMP goes down, and vice versa

what genes does the trp operon control?

genes for tryptophan synthesis (before being attached to tRNA)

what is the corepressor for the trp operon?

tryptophan (its own product) - makes negative feedback to prevent excessive Trp synthesis

how does tryptophan interact with its repressor?

the repressor will be inactive, and the corepressor will bind to activate the repressor and stop transcription - the operon will cycle and activate/deactivate itself as an initiation control

what kind of interaction does the trp promoter have?

negative repressible

what two types of proteins do the Trp operons encode?

enzymes involved in synthesizing tryptophan and proteins that make up the system repressor

what is the summary pathway for the trp operon initiation control?

in low trp, repressor is not effective, which produces synthesis enzymes, which makes trp levels increase, which will act as corepressors and bind to the repressors, which will repress the operon and reduce trp synthesis

what two pools of tryptophans is the trp operon under dual control from?

free tryptophan, for the promoter repression, and tRNA-Trp, for the termination repression

how does the cell respond to tRNA-Trp?

through attenuation - it’s not enough to completely repress operon expression, but it can reduce it

how does the operon sense the level of tRNA-Trp?

needs a protein complex (ribosome!) that will bind to tRNA-Trp with high affinity; therefore, tRNA levels are sensed through translation process, as low tRNA levels results in ribosome stalling

what is the mechanism for the Trp operon attenuation?

cell will take advantage of transcription and translation properties, so the TRP operon developed a special sequence (leader sequence - between operator and first gene) - attenuator is the terminator for that sequence, and if that stem loop terminator’s used, transcription is prevented

how does the cell decide whether to use the regular terminator or the attenuation terminator?

if there are high tRNA-Trp levels, use attenuator terminator; if low tRNA-Trp levels, use regular terminator

how does the attenuator respond to tRNA-TRp?

the leader sequence allows for two alternative base-pairings in its transcript; 2:3 (acts as antiterminator, so termination can’t be triggered) and 1:2&3:4 (acts as terminator). when tRNA-Trp levels are high, ribosome affects 2:3 pairing, so 1:2&3:4 pairing occurs and transcription stops; when those levels are low, ribosome only allows 2:3 pairing, which allows transcription to continue