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MBB164 > Barnes (Gene Expression) > Flashcards

Barnes (Gene Expression) Flashcards

1
Q

What is gene expression?

A
  • product being transcribed and translated
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2
Q

Does E. coli express all its proteins at once, why?

A
  • only ~700 of 4000 proteins present in cell at any 1 time

- as proteins needed depends on env conditions

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3
Q

Why is regulation necessary?

A
  • efficiency –> waste of energy and resources if protein made and not used
  • to avoid chaos
  • allow adaption to env
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4
Q

What is constitutive expression, and an example?

A
  • always expressed
  • housekeeping genes, req for basic cell function
  • eg. transcrip enzymes
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5
Q

What is facultative/responsive/adaptive expression, and an example?

A
  • prod in response to certain stimuli
  • inducible/repressible genes (switched on/off)
  • eg. enzymes for lactose metabolism
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6
Q

What does fine control involve?

A
  • instant responses
  • alteration of critical enzymes
  • activity of enzymes and other proteins alt by covalent mod or by binding of other molecules
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7
Q

What does coarse control involve?

A
  • delayed responses
  • LT changes, +ve or -ve gene reg
  • slow but v economical
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8
Q

What are the diff types of fine control?

A
  • irreversible means of alt enzyme activity
  • reversible AA mods
  • reversible ligand binding
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9
Q

What are the diff types of reversible AA mods? (fine control)

A
  • phosphorylation
  • acetylation
  • ubiquitination
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10
Q

What is feedback inhibition? (reversible ligand binding)

A
  • reaction products interact w/ active site

- preventing further substrate binding

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11
Q

What are the diff ways coarse control can occur?

A
  • can alter rate of synthesis, or rate of degradation, or both
  • reg at many diff levels to increase or decrease amount of protein in cell
  • reg at both transcriptional and posttranscriptional levels
  • reg of transcriptional initiation most important
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12
Q

What does it mean to say genes are “pre-set”?

A
  • cell req diff amounts of each constitutively expressed proteins
  • most E. coli genes present in only 1 copy per genome
  • variable strengths of diff gene promoters and of ribosome binding sites on mRNAs
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13
Q

How is coarse control of transcrip carried out?

A
  • bacterial promoters upstream of transcrip start site, bind transcrip machinery
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14
Q

What is the role of DNA binding proteins?

A
  • reg of RNA pol binding and transcrip initiation proteins that bind specific DNA seqs around promoter
  • bind to DNA via DNA recognition sites in protein structure
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15
Q

What is the diff between +ve and -ve control DNA binding proteins?

A
  • +ve = bind and increase transcrip (activators)

- -ve = bind and decrease transcrip (repressor)

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16
Q

What affects bacterial promoter strength?

A
  • seqs closer to ideal consensus seq bind transcriptional machinery more strongly, so transcribed more
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17
Q

Why so mRNAs have short half lives?

A
  • metabolically unstable in bacteria (half lives of a few mins)
  • allow genes to be switched on/off v quickly
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18
Q

How do protein half lives compare to mRNAs?

A
  • longer (hours/days)
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19
Q

What happens during coarse control of translation?

A
  • ribosomes bind to specific sites in mRNA (SD consensus seq)
  • similarity to consensus determines efficiency of ribosome binding and translation
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20
Q

What are the characteristics of bacterial operons?

A
  • several protein coding genes
  • single promoter
  • regulatory seq in region of promoter is operator
  • regulatory regions bound by reg proteins to repress or activate transcrip depending on conditions
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21
Q

What is the role of regulatory proteins?

A
  • bind specific ligands to determine concs
  • then bind to DNA regulatory seq and mod transcrip rate
  • binding to operator seq down regs expression
  • binding to activator increases expression
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22
Q

What is polycistronic transcription?

A
  • many genes in a single transcript
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23
Q

What are the main features of the lac operon?

A
  • structural genes (= protein coding)
  • single promoter
  • regulatory regions around promoter
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24
Q

What is the activity of regulatory proteins affected by, and how?

A
  • binding of various small molecules, prob metabolites

- act as signal telling regulatory protein whether to bind to regulatory region, by changing conformation

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25
Q

What does binding of regulatory protein do under -ve control and where does it bind?

A
  • inhibits expression

- operator seq

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26
Q

What does binding of regulatory protein do under +ve control and where does it bind?

A
  • enhances expression

- activator seq

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27
Q

What C source do bacteria prefer to use, and why?

A
  • glucose
  • most efficient way to get energy
  • can use alt when glucose scarce
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28
Q

Why don’t bacteria express genes to metabolise all sugars all the time?

A
  • transcrip and translation costly to cell

- converting other sugars to a form allowing them to enter biochem pathway also costly

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29
Q

What do the diff genes of the lac operon code for?

A
  • LacZ = β-galactosidase (breaks down lactose)
  • LacY = lactose permease (transports lactose into cell
  • LacA = thiogalactosidase transacetylase
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30
Q

What reaction does β-galactosidase catalyse?

A
  • lactose + water –> galactose + glucose

- galactose converted to glucose

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31
Q

In what situation does E. coli want the lac operon to be expressed?

A
  • lactose available

- low glucose

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32
Q

How does bacterial cell control expression of lac operon genes?

A
  • -ve control = LacI inhibits expression when not req

- +ve control = CRP activates expression when req

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33
Q

What gene is β-galactosidase related to humans?

A
  • lactase
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34
Q

What is the role of repressor LacI in -ve control?

A
  • binds to symmetrical operator seq, blocking transcrip
  • constitutively expressed
  • forms tetramer w/ 4 LacI and and binds 2 LacI binding sites at once forming DNA loop
  • preventing RNA pol from transcribing genes
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35
Q

Where is the repressor LacI expressed in E. coli and is this always the case?

A
  • just upstream w/ own promoter

- no, coincidence

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36
Q

How is allolactose made in the lac operon?

A
  • β-galactosidase converts small proportion of lactose to allolactose
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37
Q

What is the role of allolactose in the lac operon, when lactose is present?

A
  • binds to LacI, changing protein shape so it can’t bind to operator seq
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38
Q

In summary what happens when lactose is absent?

A
  • operon not needed
  • repressor binds operator
  • no transcrip
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39
Q

In summary what happens when lactose is present?

A
  • operon needed
  • repressor doesn’t bind operator
  • transcrip proceeds
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40
Q

What does it mean to say the lac operon is incompletely repressed?

A
  • always tiny bit of expression
  • less than 5 β-galactosidase molecules per uninduced cell
  • β-galactosidase req for repression of own expression
41
Q

What is diauxic growth?

A
  • “double growth”

- cell uses up glucose, then lactose

42
Q

How are glucose levels communicated?

A
  • cAMP levels

- adenylate cyclase converts ATP –> cAMP

43
Q

What happens in relation to adenylate cyclase activity at diff glucose concs?

A
  • high glucose = low adenylate cyclase activity, low cAMP conc
  • low glucose = high adenylate cyclase activity, high cAMP conc
44
Q

How is amount of glucose in env related to amount transported into cell?

A
  • inversely proportional
45
Q

What is the role of CRP (cAMP receptor protein) / CAP?

A
  • forms homodimer and binds to specific DNA seq
  • binding activates transcrip (recruitment of RNA pol)
  • but only when assoc w/ cAMP (conformational change allowing it to bind DNA)
46
Q

Where is the CRP binding site seq and what does it contain?

A
  • upstream of range of genes involved in metabolism of non-glucose sugars
  • binding site contains inverted repeats
47
Q

What happens to CRP when there is high glucose and low cAMP?

A
  • CRP in conformation that can’t bind to binding site at lac promoter
48
Q

What happens to CRP when there is low glucose and high cAMP

A
  • cAMP binds CRP and changes its conformation

- CRP activates transcrip

49
Q

What is basal transcrip of the lac operon?

A
  • lactose high, LacI doesn’t bind

- glucose high, CRP doesn’t bind

50
Q

In what situation is there no expression of the lac operon?

A
  • lactose low, LacI binds

- glucose high, CRP doesn’t bind

51
Q

How can the lac operon be used to express a protein in large quantities in the lac operon?

A
  • intro DNA seq containing lac promoter upstream of foreign genes into E. coli genome
  • bacteria has own lac operon and expresses lac repressor protein
  • IPTG binds to repressor in same way as allolactose normally does, allowing strong transcrip factor from this promoter
  • IPTG not broken down by β-galactosidase, so lasts longer than lactose would
52
Q

What is the role of IPTG?

A
  • binds lac repressor instead of allolactose and prevents binding to operator seq
53
Q

What is the role of the ara operon?

A
  • encodes genes involved in arabinose metabolism (a 5C carb)
  • arabinose converted to xylulose 5-phosphate
  • proteins to transport arabinose into cell expressed separately
54
Q

What is the role of the ara operon in the pentose phosphate pathway?

A
  • preferred pathway uses xylulose 5-phosphate

- arabinose can be used instead of glucose to feed into pathway there

55
Q

What happens at diff glucose concs in +ve reg of ara operon?

A
  • low glucose, high cAMP = cAMP binds to CRP and changes conformation, CRP binds DNA, transcrip of operon, arabinose metabolised
  • high glucose, low cAMP = cAMP in non-binding conformation, CRP doesn’t bind DNA, operon not transcribes, arabinose not metabolised
56
Q

How is the ara operon repressed/activated?

A
  • AraC is repressor and activator
  • “light switch model”
  • arabinose binding changes position of AraC DNA binding domain
  • can form dimers in 2 diff conformations
  • AraC dimer binds to I1 and to ether I2 or O2
57
Q

What are the 2 diff conformations that AraC can form as a dimer?

A
  • when arabinose bound, DNA binding domains side by side

- when arabinose not bound, DNA binding domain apart

58
Q

What happens in when no arabinose present?

A
  • -ve reg

- AraC binds so DNA is looped and no transcrip can occur

59
Q

What happens when arabinose present?

A
    • reg
  • binds to AraC protein and AraC binds DNA
  • transcrip proceeds
60
Q

What does bacteriophage λ infect?

A
  • E. coli
61
Q

What are the characteristics of the bacteriophage λ genome?

A
  • ds DNA genome
  • 48kb
  • contains genes for diff viral components and for control of lysis and lysogeny
62
Q

What happens during lysis?

A
  • ends in destruction of host cell
  • viral DNA rep separately from host genome
  • gene products needed for lysis = proteins to make phage parts and to initiate rep of viral DNA
  • most genome made up of proteins needed for lytic cycle
63
Q

What happens during lysogeny?

A
  • “temperate lifestyle”
  • host cell can still function normally
  • viral DNA integrated into host genome (= prophage)
  • most viral genes not expressed
64
Q

How do cloudy/clear plaques show whether in lysogeny or lytic life cycle?

A
  • lawn of E. coli infected w/ bacteriophage λ
  • plaques form, corresponding to infected cells
  • cloudy = lysogenic, growth slowed
  • clear = lytic, bacteria burst open
65
Q

What are the regulatory proteins of the lytic cycle, and their role?

A
  • Cro = binds to DNA to control transcrip
  • N = antitermination factor, allows transcrip to proceed
  • Q = antitermination factor, allows transcrip to proceed
  • N and Q both bind to nascent RNA and change 2º structure
66
Q

What are the regulatory proteins of the lysogenic cycle, and their role?

A
  • CI = binds DNA to control transcrip (competing w/ Cro)

- CII and CIII reg CI function

67
Q

What are the 4 promoters, their genes and the gene function?

A
  • PL –> N –> lysis
  • PR –> Cro, Q, O, P –> lysis control, lytic rep
  • PR’ –> phage particle components –> lysis
  • PRM –> CI –> repression of lytic promoters
68
Q

How is the operator region in lytic/lysogenic cycle?

A
  • OL at PL
  • OR at PR
  • each operator contains 3 binding sites for Cro or CI, each 17bp
  • slight differences between repeats, each w/ slightly diff affinities for Cro and CI
  • overlap between operator and transcrip start seqs
  • so binding of proteins to operator can prevent transcrip
  • only 1 protein can be bound at 1 time
69
Q

What is the lytic life cycle controlled by?

A
  • expression of Cro and N proteins from PR and PL respectively
70
Q

Where does Cro bind w/ the highest affinity, and what does this cause?

A
  • operator region 3
  • at OR, binding of of Cro to region 3 blocks transcrip from PRM, but doesn’t affect PR
  • at OL, doesn’t affect transcrip from PL
71
Q

What is expressed when Cro bound to operators?

A
  • Cro and N
72
Q

What is the role of the antitermination factor N in the lytic pathway?

A
  • allows expression of distal genes from PR and PL

- removes 2º structure from new RNA that would otherwise terminate transcrip

73
Q

What is the role of the antitermination factor Q in the lytic pathway?

A
  • allows expression of distal genes from PR

- removes 2ºstructure from RNA that would otherwise terminate transcrip

74
Q

In summary, how is the lytic cycle controlled?

A
  • Cro binds to OL and OR, allowing expression of PL
    and PR
  • N allows these transcripts to be extended
  • O and P rep viral DNA
  • Q extends transcription from PR’ –> expression of other
    proteins that make up infectious viral particle
75
Q

What is the only protein expressed by lysogenic phage, and what does it do?

A
  • CI

- represses everything else

76
Q

Where does CI bind w/ the highest affinity, and what does this cause?

A
  • operator regions 1 and 2
  • at OR, binding of CI to regions 1 and 2 blocks transcrip from PR, but doesn’t affect transcrip from PRM
  • at OL, binding of CI protein to regions 1 and 2 blocks transcrip from PL
77
Q

What is basal induction, and what can cause it?

A
  • induction of lysogenic prophage to enter lytic cycle
  • 1/1000 chance of spontaneous activation of prophage
  • or by DNA damage
78
Q

What is the series of events following starvation or severe DNA damage?

A
  • lots of effects inc activation of certain protease
  • cleavage of CI
  • dissociation of CI from OL and OR
  • transcrip from PL and PR
  • expression of lytic genes
79
Q

How is decision between lysis and lysogeny made?

A
  • competition between Cro and CI to bind to OL and OR
80
Q

When is lytic cycle activated on entry into new cell?

A
  • Cro “wins”, bound to OR and OL

- expression from PR and PL

81
Q

When is lysis likely to happen over lysogeny?

A
  • low multiplicity of infection
  • rich media
  • severe DNA damage
82
Q

When is lysogenic cycle activated upon entry into new cell?

A
  • CI “wins”, bound to OR and OL
  • expression from PRM (or PRE)
  • no expression from PR and PL
83
Q

When is lysogeny likely to happen over lysis?

A
  • high multiplicity of infection

- poor media

84
Q

What are the phases of phage gene expression on entry into new bacterial cell?

A
  • early transcrip (nothing bound to operator) = just Cro and N expressed
  • delayed early transcrip = both lytic (Cro, N) and lysogenic (CII) proteins expressed from PL and PR
  • balance between lytic (Cro) and lysogenic (CI) protein levels determines path taken
85
Q

What promoters can CI be expressed from and what are they involved in?

A
  • PRM –> repression maintenance, activated by CI

- PRE –> repression establishment,activated by CII

86
Q

How does CII promote lysogeny?

A
  • by activating P1 and PRE
87
Q

What is the role of PRE?

A
  • CII made from rightward transcript (PR) during delayed early phase of infection
  • CII induces synthesis of CI from PRE
  • CI binds to OR and OL, blocks transcrip of lytic genes from PR and PL
  • high CII –> high CI –> lysogeny
88
Q

When is CII only expressed?

A
  • during establishment of lysogeny
89
Q

What is the multiplicity of infection?

A
  • ratio of no. of virus particles to no. of target cell present in defined space
  • high MOI = many infections per bacterial cell
90
Q

How does MOI affect lysis?

A
  • if MOI low, surrounding cells prob not already infected

- in viruses interests to lyse cell and spread to surrounding uninfected cells

91
Q

How does MOI affect lysogeny?

A
  • if MOI high, surrounding cells prob already infected

- in viruses interests to enter lysogeny, so doesn’t wipe out entire pop of pot hosts

92
Q

How do nutrient levels affect lysis?

A
  • if glucose levels high, surrounding cells prob growing healthily
  • in viruses interests to lyse cell and spread to surrounding uninfected cells
  • in rich media, high levels of N antitermination factor = lysis
93
Q

How do nutrient levels affect lysogeny?

A
  • if glucose levels low, surrounding cells prob unhealthy
  • in viruses interests to enter lysogeny, so doesn’t wipe out entire pop of pot hosts
  • in poor media, low levels of N antitermination factor = lysogeny
94
Q

How are CI levels controlled by CII/CIII/FtsH?

A
  • CI represses lytic promoters = lysogeny
  • CII induces expression of CI from PRE
  • high CII –> high CI –> lysogeny
  • low CII –> low CI –> lysis
  • FtsH protease degrades CII
  • CIII blocks FtsH protease activity
95
Q

How does high MOI / low glucose lead to lysogeny?

A
  • repression of FtsH
  • high levels of CII
  • expression of CI
  • CI binds to OR and OL
  • expression of PRM but not PR or PL
96
Q

How does low MOI / high glucose lead to lysis?

A
  • activity of FtsH
  • low levels of CII
  • CI is not expressed from PRE
  • Cro binds to OR and OL
  • Expression of PR and PL, but not PRM
97
Q

In summary, how is lysis established?

A
  • Cro and N expressed in early transcrip
  • low MOI and/or high glucose
  • FtsH degrades CII
  • low CII protein levels
    CI is not expressed from PRE
    Cro binds to OR and OL without competition from CI
98
Q

In summary, how is lysogeny established?

A
  • Cro and N expressed in early transcrip
  • high MOI and/or low glucose
    FtsH activity blocked
  • CII accumulates above threshold level
  • CII binds to PRE
  • CI expressed from PRE
  • CI binds to OR and OL, beats Cro = silencing of PR and PL

MBB164 (7 decks)

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