Barnes - Gene expression

Question 1

why do prokaryotes regulate genes?

Answer 1

efficiency - doesnt waste energy and resources
avoids chaos
adaption - cells adapt to env

Question 2

name the 2 types of gene expression and explain them

Answer 2

constitutive: constant expression
facultative: selective expression

Question 3

define fine and coarse control

Answer 3

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

Question 4

describe methods of reversible and irreversible fine control

Answer 4

irreversible: altering enzyme activity
reversible: a/a modification eg phosphorylation
ligand binding - allosterism/feedback inhibition

Question 5

does coarse control act on transcription, translation or both in prokaryotes?

Answer 5

both, because they are coupled

Question 6

name 2 regulatory proteins and state the difference in control method and where they bind

Answer 6

Repressor
- Negative control
- Binds operator
Activator
- Positive control
- Binds control elements eg regulatory sites near promoter or enhancer regions

Question 7

state the promoter region place in e. coli

Answer 7

35 and 10 NTPs upstream transc start site in E. coli

Question 8

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

Answer 8

Closer to consensus seq the promoter is, the stronger the transcription machinery binds to the DNA
Closer to SD sequence = increased translation (coarse control)

Question 9

what property of mRNA means genes can be switched on/off quickly?

Answer 9

mRNA is metabolically unstable in bacteria and has a short 1/2 life - can respond to cellular changes more quickly

Question 10

what is the SD (shine dalgarno) sequence

Answer 10

UAAGGAGG

Question 11

define polycistronic transcription

Answer 11

the arrangement of several protein encoding genes needed in similar circumstances that can be transcribed and translated together

Question 12

how does e. coli use glucose?

Answer 12

glucose –> pyruvate –> energy

Question 13

name the 3 structural genes in e. coli’s lac operon and describe what they do

Answer 13

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?)

Question 14

describe the negative control mechanism in the lac operon

Answer 14

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

Question 15

describe the positive control mechanism in the lac operon

Answer 15

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

Question 16

what is IPTG? why is it used?

Answer 16

IPTG = allolactose substitute (not broken down by beta-galactosidase)
used in research to produce a protein of interest

Question 17

state which molecules are involved in the positive and negative control mechanisms in the ara operon

Answer 17

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

Question 18

what do clear and cloudy plaques on a lawn of E. coli indicate about the lifecycle bacteriophage lambda?

Answer 18

clear plaque: lytic - clear because the phage has lysed the cells (they’re empty)
cloudy: lysogenic - E. coli cells havent been lysed

Question 19

name the 3 lytic cycle regulators in bacteriophage λ and state what they do

Answer 19

cro: binds to DNA to control transcription
N: antitermination factor - allows transcription to carry on
Q: antitermination factor - allows transcription to carry on

Question 20

name the 3 lysogenic cycle regulators in bacteriophage λ and state what they do

Answer 20

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

Question 21

what do genes O and P do?

Answer 21

carry out replication of the viral DNA

Question 22

how does the binding of proteins to the operator regions prevent transcription from the promoters?

Answer 22

the operator regions overlap with the transcriptional start sequences therefore a protein binding to the operator can prevent transcription

Question 23

which region in the operators does Cro have the highest affinity for? what does this mean for transcription at the promoters?

Answer 23

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)

Question 24

how does N work as an antitermination factor?

Answer 24

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

Question 25

how does Q work as an antitermination factor?

Answer 25

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

Question 26

which region in the operators does CI have the highest affinity for? what does this mean for transcription at the promoters?

Answer 26

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)

Question 27

in what 3 cases will a lysogenic prophage enter the lytic cycle?

Answer 27

1) 1/1000 spontaneous activation of prophage 'basal induction' 2) DNA damage eg UV light in a lab 3) extreme starvation

Question 28

describe in brief how a lysogenic prophage enters the lytic cycle

Answer 28

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

Question 29

how does a phage ''decide'' to enter lysis or lysogeny upon entering a new bacterial cell?

Answer 29

``` 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 ```

Question 30

define multiplicity of infection (MOI)

Answer 30

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

Question 31

name and describe the 2 phases of gene expression upon entry into a new bacterial cell

Answer 31

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

Question 32

describe how lysis is established when phage λ has entered a new bacterial cell

Answer 32

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

Question 33

describe how lysogeny is established when phage λ enters a new bacterial cell

Answer 33

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

Question 34

describe the maintenance of lysis in phage λ

Answer 34

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

Question 35

describe the maintenance of lysogeny in phage λ

Answer 35

CI bound to OR and OL from lysogeny establishment - silencing of PR and PL and expression PRM CI expressed from PRM