Barnes - Gene expression Flashcards
why do prokaryotes regulate genes?
efficiency - doesnt waste energy and resources
avoids chaos
adaption - cells adapt to env
name the 2 types of gene expression and explain them
constitutive: constant expression
facultative: selective expression
define fine and coarse control
fine = instant response/alterations of critical enzymes eg not making them or destroying them (achieved by covalent modification/binding of ligands)
coarse = delayed response (long-term changes +ve/-ve gene regulation
slow and economical - total cellular population of enzymes changed
describe methods of reversible and irreversible fine control
irreversible: altering enzyme activity
reversible: a/a modification eg phosphorylation
ligand binding - allosterism/feedback inhibition
does coarse control act on transcription, translation or both in prokaryotes?
both, because they are coupled
name 2 regulatory proteins and state the difference in control method and where they bind
Repressor - Negative control - Binds operator Activator - Positive control - Binds control elements eg regulatory sites near promoter or enhancer regions
state the promoter region place in e. coli
35 and 10 NTPs upstream transc start site in E. coli
describe the relationship between the consensus sequence and the promoter sequence and explain how this is similar to the shine dalgarno sequence (SD seq) in translation
Closer to consensus seq the promoter is, the stronger the transcription machinery binds to the DNA
Closer to SD sequence = increased translation (coarse control)
what property of mRNA means genes can be switched on/off quickly?
mRNA is metabolically unstable in bacteria and has a short 1/2 life - can respond to cellular changes more quickly
what is the SD (shine dalgarno) sequence
UAAGGAGG
define polycistronic transcription
the arrangement of several protein encoding genes needed in similar circumstances that can be transcribed and translated together
how does e. coli use glucose?
glucose –> pyruvate –> energy
name the 3 structural genes in e. coli’s lac operon and describe what they do
LacZ: beta-galactosidase (lactose to glucose and galactose as well as lactose –> allolactose)
LacY: lactose permease (transports lactose into the cell)
LacA: thiogalactoside transacetylase (removes thiogalactosides - maybe involved in detox?)
describe the negative control mechanism in the lac operon
lacI gene expressed –> LacI repressor protein transc and transl (tetramer)
LacI protein binds to operator and upstream sequence –> loop –> no RNA pol activity
incomplete repression of LacZ allows some lactose –> allolactose
when lactose is present allolactose binds LacI (undergoes allosteric change, cannot bind operator, RNA pol transcribes
describe the positive control mechanism in the lac operon
adenylate cyclase (AC) activity inhibited by glucose transport when glucose conc high = AC activity low = low cAMP. CRP forms a homodimer and bind to CAP/CRP site cAMP + CRP + DNA + RNA pol = transc therefore when glucose is present there is low cAMP and CRP cant bind to the CRP binding region - no transcription of the downstream structural genes
what is IPTG? why is it used?
IPTG = allolactose substitute (not broken down by beta-galactosidase)
used in research to produce a protein of interest
state which molecules are involved in the positive and negative control mechanisms in the ara operon
positive control:
by CRP - binds CRP binding region = transc
by arabinose - binds AraC dimer protein which binds I1 and I2 = trancs
negative control:
by arabinose - when not present AraC dimer protein binds O2 and I1
what do clear and cloudy plaques on a lawn of E. coli indicate about the lifecycle bacteriophage lambda?
clear plaque: lytic - clear because the phage has lysed the cells (they’re empty)
cloudy: lysogenic - E. coli cells havent been lysed
name the 3 lytic cycle regulators in bacteriophage λ and state what they do
cro: binds to DNA to control transcription
N: antitermination factor - allows transcription to carry on
Q: antitermination factor - allows transcription to carry on
name the 3 lysogenic cycle regulators in bacteriophage λ and state what they do
CI: binds DNA to control transcription (at OR and OL)
CII: binds DNA to control lysogenic establishment (binds Pi and Pre)
CIII: inhibits FtsH activity
what do genes O and P do?
carry out replication of the viral DNA
how does the binding of proteins to the operator regions prevent transcription from the promoters?
the operator regions overlap with the transcriptional start sequences therefore a protein binding to the operator can prevent transcription
which region in the operators does Cro have the highest affinity for? what does this mean for transcription at the promoters?
Cro binds with highest affinity at region 3 of OR and OL
At OR binding of Cro to region 3 blocks transc from PRM (no CI) but not from PR (Cro expression)
At OL binding of Cro to region 3 doesnt affect transcription from PL (expression of N)
how does N work as an antitermination factor?
N allows expression off distal genes from PR and PL. N binding to nascent mRNA removes the secondary structures that would stop O, P and Q being transcribed
how does Q work as an antitermination factor?
Q allows expression of distal genes from PR’ by binding to nascent mRNA and removing secondary structures that inhibit the transc of the structural genes
which region in the operators does CI have the highest affinity for? what does this mean for transcription at the promoters?
CI binds with the highest affinity to regions 1 and 2 in the operators
at OR binding of CI to regions 1&2 blocks transc from PR (no Cro) but not from PRM (expression CI)
At OL binding of CI to regions 1&2 blocks transcription from PL (no N or CIII)
in what 3 cases will a lysogenic prophage enter the lytic cycle?
1) 1/1000 spontaneous activation of prophage ‘basal induction’
2) DNA damage eg UV light in a lab
3) extreme starvation
describe in brief how a lysogenic prophage enters the lytic cycle
1) starvation/DNA damage
2) protease breaks down CI
3) dissociation of CI from OR and OL
4) transcription from PL and PR
5) expression of the lytic genes
how does a phage ‘‘decide’’ to enter lysis or lysogeny upon entering a new bacterial cell?
lysis - Cro expression if: low multiplicity of infection (MOI) high glucose conc/rich media severe DNA damage lysogeny - CI expression if: high MOI poor media/low glucose conc
define multiplicity of infection (MOI)
the ratio of viruses per bacterial cell
eg MOI = 2: 2 viruses per 1 bacterial cell
MOI = 0.5: 1 virus per 2 bacterial cells
name and describe the 2 phases of gene expression upon entry into a new bacterial cell
early transcription:
nothing bound to the operators - only Cro and N expressed
delayed-early transcription:
both lytic (Cro and N) and lysogenic (CII) proteins are expressed from PL and PR
describe how lysis is established when phage λ has entered a new bacterial cell
Cro, N, CII expressed in delayed-early transcription
Low MOI/high glucose conc = low cAMP = FtsH not repressed therefore degrades CII
Low CII levels means there is no expression from PRE of CI
therefore Cro can bind OR and OL without competition from CI - silencing of PRM
describe how lysogeny is established when phage λ enters a new bacterial cell
Cro, N and CII expressed in delayed-early transcription
High MOI/low glucose = high cAMP = FtsH repressed = no degradation of CII
CII accumulates above threshold value
CII binds PRE
CI expressed from PRE
CI binds Or and OL before Cro - silencing of PR and PL
describe the maintenance of lysis in phage λ
Cro bound to OR and OL from establishment of lysis - PR and PL expressed
CIII expressed PL = inhibition of FtsH
N antitermination of PL
Q antitermination of PR’
expression of lytic genes and structural genes
describe the maintenance of lysogeny in phage λ
CI bound to OR and OL from lysogeny establishment - silencing of PR and PL and expression PRM
CI expressed from PRM
