Barnes (Gene Expression) Flashcards
What is gene expression?
- product being transcribed and translated
Does E. coli express all its proteins at once, why?
- only ~700 of 4000 proteins present in cell at any 1 time
- as proteins needed depends on env conditions
Why is regulation necessary?
- efficiency –> waste of energy and resources if protein made and not used
- to avoid chaos
- allow adaption to env
What is constitutive expression, and an example?
- always expressed
- housekeeping genes, req for basic cell function
- eg. transcrip enzymes
What is facultative/responsive/adaptive expression, and an example?
- prod in response to certain stimuli
- inducible/repressible genes (switched on/off)
- eg. enzymes for lactose metabolism
What does fine control involve?
- instant responses
- alteration of critical enzymes
- activity of enzymes and other proteins alt by covalent mod or by binding of other molecules
What does coarse control involve?
- delayed responses
- LT changes, +ve or -ve gene reg
- slow but v economical
What are the diff types of fine control?
- irreversible means of alt enzyme activity
- reversible AA mods
- reversible ligand binding
What are the diff types of reversible AA mods? (fine control)
- phosphorylation
- acetylation
- ubiquitination
What is feedback inhibition? (reversible ligand binding)
- reaction products interact w/ active site
- preventing further substrate binding
What are the diff ways coarse control can occur?
- 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
What does it mean to say genes are “pre-set”?
- 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
How is coarse control of transcrip carried out?
- bacterial promoters upstream of transcrip start site, bind transcrip machinery
What is the role of DNA binding proteins?
- 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
What is the diff between +ve and -ve control DNA binding proteins?
- +ve = bind and increase transcrip (activators)
- -ve = bind and decrease transcrip (repressor)
What affects bacterial promoter strength?
- seqs closer to ideal consensus seq bind transcriptional machinery more strongly, so transcribed more
Why so mRNAs have short half lives?
- metabolically unstable in bacteria (half lives of a few mins)
- allow genes to be switched on/off v quickly
How do protein half lives compare to mRNAs?
- longer (hours/days)
What happens during coarse control of translation?
- ribosomes bind to specific sites in mRNA (SD consensus seq)
- similarity to consensus determines efficiency of ribosome binding and translation
What are the characteristics of bacterial operons?
- 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
What is the role of regulatory proteins?
- 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
What is polycistronic transcription?
- many genes in a single transcript
What are the main features of the lac operon?
- structural genes (= protein coding)
- single promoter
- regulatory regions around promoter
What is the activity of regulatory proteins affected by, and how?
- binding of various small molecules, prob metabolites
- act as signal telling regulatory protein whether to bind to regulatory region, by changing conformation
What does binding of regulatory protein do under -ve control and where does it bind?
- inhibits expression
- operator seq
What does binding of regulatory protein do under +ve control and where does it bind?
- enhances expression
- activator seq
What C source do bacteria prefer to use, and why?
- glucose
- most efficient way to get energy
- can use alt when glucose scarce
Why don’t bacteria express genes to metabolise all sugars all the time?
- transcrip and translation costly to cell
- converting other sugars to a form allowing them to enter biochem pathway also costly
What do the diff genes of the lac operon code for?
- LacZ = β-galactosidase (breaks down lactose)
- LacY = lactose permease (transports lactose into cell
- LacA = thiogalactosidase transacetylase
What reaction does β-galactosidase catalyse?
- lactose + water –> galactose + glucose
- galactose converted to glucose
In what situation does E. coli want the lac operon to be expressed?
- lactose available
- low glucose
How does bacterial cell control expression of lac operon genes?
- -ve control = LacI inhibits expression when not req
- +ve control = CRP activates expression when req
What gene is β-galactosidase related to humans?
- lactase
What is the role of repressor LacI in -ve control?
- 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
Where is the repressor LacI expressed in E. coli and is this always the case?
- just upstream w/ own promoter
- no, coincidence
How is allolactose made in the lac operon?
- β-galactosidase converts small proportion of lactose to allolactose
What is the role of allolactose in the lac operon, when lactose is present?
- binds to LacI, changing protein shape so it can’t bind to operator seq
In summary what happens when lactose is absent?
- operon not needed
- repressor binds operator
- no transcrip
In summary what happens when lactose is present?
- operon needed
- repressor doesn’t bind operator
- transcrip proceeds
What does it mean to say the lac operon is incompletely repressed?
- always tiny bit of expression
- less than 5 β-galactosidase molecules per uninduced cell
- β-galactosidase req for repression of own expression
What is diauxic growth?
- “double growth”
- cell uses up glucose, then lactose
How are glucose levels communicated?
- cAMP levels
- adenylate cyclase converts ATP –> cAMP
What happens in relation to adenylate cyclase activity at diff glucose concs?
- high glucose = low adenylate cyclase activity, low cAMP conc
- low glucose = high adenylate cyclase activity, high cAMP conc
How is amount of glucose in env related to amount transported into cell?
- inversely proportional
What is the role of CRP (cAMP receptor protein) / CAP?
- 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)
Where is the CRP binding site seq and what does it contain?
- upstream of range of genes involved in metabolism of non-glucose sugars
- binding site contains inverted repeats
What happens to CRP when there is high glucose and low cAMP?
- CRP in conformation that can’t bind to binding site at lac promoter
What happens to CRP when there is low glucose and high cAMP
- cAMP binds CRP and changes its conformation
- CRP activates transcrip
What is basal transcrip of the lac operon?
- lactose high, LacI doesn’t bind
- glucose high, CRP doesn’t bind
In what situation is there no expression of the lac operon?
- lactose low, LacI binds
- glucose high, CRP doesn’t bind
How can the lac operon be used to express a protein in large quantities in the lac operon?
- 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
What is the role of IPTG?
- binds lac repressor instead of allolactose and prevents binding to operator seq
What is the role of the ara operon?
- encodes genes involved in arabinose metabolism (a 5C carb)
- arabinose converted to xylulose 5-phosphate
- proteins to transport arabinose into cell expressed separately
What is the role of the ara operon in the pentose phosphate pathway?
- preferred pathway uses xylulose 5-phosphate
- arabinose can be used instead of glucose to feed into pathway there
What happens at diff glucose concs in +ve reg of ara operon?
- 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
How is the ara operon repressed/activated?
- 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
What are the 2 diff conformations that AraC can form as a dimer?
- when arabinose bound, DNA binding domains side by side
- when arabinose not bound, DNA binding domain apart
What happens in when no arabinose present?
- -ve reg
- AraC binds so DNA is looped and no transcrip can occur
What happens when arabinose present?
- reg
- binds to AraC protein and AraC binds DNA
- transcrip proceeds
What does bacteriophage λ infect?
- E. coli
What are the characteristics of the bacteriophage λ genome?
- ds DNA genome
- 48kb
- contains genes for diff viral components and for control of lysis and lysogeny
What happens during lysis?
- 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
What happens during lysogeny?
- “temperate lifestyle”
- host cell can still function normally
- viral DNA integrated into host genome (= prophage)
- most viral genes not expressed
How do cloudy/clear plaques show whether in lysogeny or lytic life cycle?
- lawn of E. coli infected w/ bacteriophage λ
- plaques form, corresponding to infected cells
- cloudy = lysogenic, growth slowed
- clear = lytic, bacteria burst open
What are the regulatory proteins of the lytic cycle, and their role?
- 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
What are the regulatory proteins of the lysogenic cycle, and their role?
- CI = binds DNA to control transcrip (competing w/ Cro)
- CII and CIII reg CI function
What are the 4 promoters, their genes and the gene function?
- PL –> N –> lysis
- PR –> Cro, Q, O, P –> lysis control, lytic rep
- PR’ –> phage particle components –> lysis
- PRM –> CI –> repression of lytic promoters
How is the operator region in lytic/lysogenic cycle?
- 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
What is the lytic life cycle controlled by?
- expression of Cro and N proteins from PR and PL respectively
Where does Cro bind w/ the highest affinity, and what does this cause?
- 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
What is expressed when Cro bound to operators?
- Cro and N
What is the role of the antitermination factor N in the lytic pathway?
- allows expression of distal genes from PR and PL
- removes 2º structure from new RNA that would otherwise terminate transcrip
What is the role of the antitermination factor Q in the lytic pathway?
- allows expression of distal genes from PR
- removes 2ºstructure from RNA that would otherwise terminate transcrip
In summary, how is the lytic cycle controlled?
- 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
What is the only protein expressed by lysogenic phage, and what does it do?
- CI
- represses everything else
Where does CI bind w/ the highest affinity, and what does this cause?
- 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
What is basal induction, and what can cause it?
- induction of lysogenic prophage to enter lytic cycle
- 1/1000 chance of spontaneous activation of prophage
- or by DNA damage
What is the series of events following starvation or severe DNA damage?
- 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
How is decision between lysis and lysogeny made?
- competition between Cro and CI to bind to OL and OR
When is lytic cycle activated on entry into new cell?
- Cro “wins”, bound to OR and OL
- expression from PR and PL
When is lysis likely to happen over lysogeny?
- low multiplicity of infection
- rich media
- severe DNA damage
When is lysogenic cycle activated upon entry into new cell?
- CI “wins”, bound to OR and OL
- expression from PRM (or PRE)
- no expression from PR and PL
When is lysogeny likely to happen over lysis?
- high multiplicity of infection
- poor media
What are the phases of phage gene expression on entry into new bacterial cell?
- 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
What promoters can CI be expressed from and what are they involved in?
- PRM –> repression maintenance, activated by CI
- PRE –> repression establishment,activated by CII
How does CII promote lysogeny?
- by activating P1 and PRE
What is the role of PRE?
- 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
When is CII only expressed?
- during establishment of lysogeny
What is the multiplicity of infection?
- ratio of no. of virus particles to no. of target cell present in defined space
- high MOI = many infections per bacterial cell
How does MOI affect lysis?
- if MOI low, surrounding cells prob not already infected
- in viruses interests to lyse cell and spread to surrounding uninfected cells
How does MOI affect lysogeny?
- if MOI high, surrounding cells prob already infected
- in viruses interests to enter lysogeny, so doesn’t wipe out entire pop of pot hosts
How do nutrient levels affect lysis?
- 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
How do nutrient levels affect lysogeny?
- 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
How are CI levels controlled by CII/CIII/FtsH?
- 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
How does high MOI / low glucose lead to lysogeny?
- repression of FtsH
- high levels of CII
- expression of CI
- CI binds to OR and OL
- expression of PRM but not PR or PL
How does low MOI / high glucose lead to lysis?
- 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
In summary, how is lysis established?
- 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
In summary, how is lysogeny established?
- 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
