Mechanism and Control of Gene Transcription I Flashcards
1
Q
Regulators of transcription
A
- TF
- Regulate expression of genes +vely and -vely
- Act primarily to control transcription initiation
- E.g. = sigma factors, activators - Nc- RNA
- Regulate expression of genes +vely/-vely
- E.g. = ribosensors/switches - DNA topology
- Signals for regulation are often environmental
2
Q
DNA topology
A
- Operon
RBS/Shine delgarno mean can still have various levels of translation e.g. LacZ, 5’UTR can be long + form structures, can terminate transcription before reach structural gene - Nucleoid
IHF bends DNA, bacterial basal expression occurs throughout the nucleiod, loop-like structure helps Pol find promoter - Toposiomerase
Bacterial chromosome = -vely supercoiled, +ve supercoils relax DNA, changes either improve or worsen
3
Q
How RNAP finds promoter
A
- Promoter search → conformational change from closed to open → abortive initiation → promoter escape
4
Q
RNA pol structure
A
- a = regulatory subunit
- B and B’ form active site, similar to RBP1 + 2 in eukaryotes
- σ = in holoenzyme, needed for initiation
- Also have w
5
Q
Sigma factors
A
- Different promoters have different factors
- 2 main classes: σ54 + σ70
- σ70 = binding at -10, -35
- σ54 binds at -24, -12
6
Q
RNA pol binding to promoter
A
- E binds to DNA randomly, slides to promoter via loop
- Kb = binding of E to DNA, sequence + structure, -10 particularly important
- K2 = melting
- At least 4 complexes formed before initiation
- -10 + -35 = recognised by 2 HTH In σ2+4
- σ1.1 prevents initiation, electrostatic interaction
- σ3.2 loops into RNAP, stab binding of initiating nucleotide substrate
7
Q
Conformational change
A
- Melting at promoter
- Template strand moves into AS, 70A moves
- Non-template captured in σ2
- ss bs interact w/ DNA → ↑ E bind
- σ2 makes interactions in prinbow box, ↑ interactions
- Torsional stress of bending => melting
8
Q
Other factors
A
- ECF σ factor
Part of σ70, -10 specific, modulate response to environmental condition, most co-transcribed w/ operons - Anti-signa factor
ASF = TM protein that binds to + inhibits cognate σ factor
9
Q
σ70 vs σ54
A
- Both have RNAP core binding domain + DBD recognising -35 or -24 elements
- 60% of bacteria genes = σ54
- Both have domain that inhibits transcription (σ1.1 in 70, R1 54)
- Both have domains that contact RNAP
- σ54 dissociates but σ70 remains loosely associated
10
Q
σ54 melting
A
- x melt DNA unlike σ70 (no K2)
- In initial inhibited state, σ54 blocks template DNA from entering RNAP AS
- Enhancer binding proteins
11
Q
bEBP
A
- Originally bind as a dimer, nucleotides alter olig state → hexamer
- Bind UAS
- Bend IHF = 180o
- Closed → open, ATP hydrolysis
- Evidence = mutations
- Causes melting at =12, brings origin of DNA melting near AS
12
Q
σ70 Activators (CRP/CAP)
A
- Need activators if σ70 has poor consensus
- E.g. Lac operon has non-consensus -35/-10, needs activator CAP/CRP
- Improve Kb by providing ↑ contacts for RNAP + K2 by further distorting NDA bend
- Structure (homodimer, 45kDa, NTD involved in dimerisation + cAMP binding, CTD has HTH, interacts w/ CTD of RNAP
Class 1 - single CRP us of -35, bs of CRP needed on same face of DNA as E, RNAP interacts w/ AR1
Class 2 = single CRP site replaces -35 on RNAP recognition region
13
Q
σ70 activator
Experiment
A
- Oriented heterodimers, identify Crp, mutant + wt Crp, co-express (us WT 1/2, ds mutant 1/2, inactive), switch = active, so ds region needed to contact RNAP
- Mutate DNA in operon + screen bacteria that induce operon in presence of glucose + induce, lacUV5 promoter
14
Q
FNR
A
- Global transcription response upon O2 deprivation
- Dimer in absence of O2, sensed through [4Fe-4S]
- O2 inactivates FNR
- Class I FNR bs = -61.5 or further us, allows contact w/ AR1 ds subunit
- Class II = FNR bs is 41.5 bp us, makes ↑ contacts w/ RNAP
15
Q
Regulating transcription
Repressors
A
- Lac operon produces proteins that bacteria metabolises to lactose
- B-galactosidase = easy to assay, link colour to it
- Agar + x-gal = colourless, B- galactosidase = blue, white = lacz-
16
Q
Lac repressor structure
A
- 3 domains: HTH, C-terminal tetrameric core, C terminus
- Binding of inducer causes a switch in N + C subdomains → closure of induce binding pocket, ↓ DNA binding
17
Q
Repression in lac operon
A
- Repressor binds LacO ds of mRNA start site
- Interferes w/ binding
- Pseudo operators (O2 40bp ds of O1, O3)
- Experiments e.g. disrupt O2 or O3 ↓ repression
- Bs = imperfect palindrome, want ↑ affinity for repressor but to fall off when induce
- DNA looping important for repressor
- LacI binds O1 + O2
18
Q
Experiment J+ M
A
- Jacob + Monod early
- Lac- mutants isolate
- 3 different substrates - J + M PAJA MA
- Used WT w/ F- recipient
- Forms merozygote, recombine DNA in F+ or F- genome
- E synthesis occurs for 30 mins w/o inducer
- Synthesis = repressed + becomes normally inducibl
19
Q
Principles from J+M experiment
A
- Regulator genes differ from structural genes e.g. lacI/ trpR vs operon
- Each regulator gene encodes a specific repressor
- In inducible, aporepressor x active when ligand bound
- In repressible system, apoprepressor is active when ligand bound
- Trp corepressor binds gene D
- Attenuation occurs
20
Q
Experiment
LacI- vs LacO-
A
- Expression from LacZ can result from 2 diff mutations
- Transacting mutations = diff gene (lacI+ = trans dominant)
- Cis-aciting = same (lacO = cis-dominant)
- Mate WT F’i+ w/ F-i+z+y+oc → constitutive
21
Q
Initiation to elongation
A
- DNA bent by almost 90o
- σ3.2 flips out
- Promoter escape by destabilising interactions btw σ4 + B flap
- Release σ4 from B flab destab interactions btw σ4 + -35 element
- RNAP gets out of promoter
22
Q
Abortive initiation
A
- Experiment = tether Ab w/ RNAP onto glass slide
- DNA packing ↑ strain of interaction btw DNA + E
- Contacts w/ promoter broken, E moves ds
- 10^-4 error
- Backtracking, can eliminate mis-incorporated nucleotides
- 3o or TEC contains E, DNA + nascent RNA
23
Q
Elongation mechanism
A
- Nucleotide addition + pyrophosphorylsis in RNAP AS
- RNA synthesis = nucleotide addition cycle: translocation of DNA/RNA, NTP binds i+1, form new phosphodiester bond, pyrophosphate release
- Reversible
- TL/TH = positional catalyst, during bond formation TL folded into AS
- Bridge helix kinks to block template until translocation
- TL/TH swing btw 2 alternative positions, essential for speed
- When folded, TH coordinates phosphate group on incoming NTP
- TL/TH maintain accuracy
- Transient phase, cap moves off, clamp loosens, RNA exits
24
Q
Backtracking
A
- RNAP pauses transiently + 3 decisions
- E balance from unwinding + rewinding
- Can have little E us for rewinding, ds unwinding requires more E than usual → elongation paused for longer
- OR, if bp in hybrid region = weaker than us e.g. dA:rU ds, dC:rC, DNA-RNA hybrid may gradually return us
- E follows bubble us, backtracking E → 3’ end RNA dipleped from AS
25
TF for backtrack recovery
- GreA + B are +ve elongation factors
- Mitigate pausing → reactivating arrested complexes
- Stimulate intrinsic hydrolytic cleavage of polymerase
- Enter 2nd channel of RNAP
26
Backtracking mechanism
- DNA pol III pauses when meets DNA sequence that impairs elongation
- Further backtracking hindered by Tyr
- Polyermase-intrinsic cleavage of di-nucleotide from 3' RNA end
- His/Arg in partially folded Tl/Th contact backtracked
- At pause, hybrid = weak, RNA backtracks beyond gating Tyr
- Traps RNA + trigger loop in pore, inhibits elongation
- TFIIS/Greb reactivates arrested PolII
Cleavage + release of backtracked RNA
27
Termination
1. Rho-independent (intrinsic)
- Identified at sequence level
- Step loop followed by U-rich sequence
2. Rho dependent
- Requires Rho factor, distributed over genome
28
Transcription elongation complex
- Stable TEC needs to be destabilised to terminate stability
- Polar contacts btw RNAP + RNA:DNA hybrid backbone
- H bonds to ssRNA In exit channel
- Contacts established on closure of clamp
29
Intrinsic terminator
- 2 factors neededL pausing + conformational change in TEC
- Intrinsic terminator = need C + G-rich hairpin to form 2o structure, followed by 7U residues
- Interactions of hairpin + RNA pol transiently misalign 3' end of AS in E
- Efficiency of termination varies
- E could instead pause before resuming elongation
30
Rho factor
- Rho factor = protein that binds nascent RNA + tracks along RNA
- Rho = hexametric, has translocase activity
- Binds Rho utilisation site us of termination site
- Uses helicase activity driven by ATP hydrolysis to translocate along NRA
- Rho causes Pol to release RNA
- Couples transcription + translation
- Rho needs access to RNA us of transcription complex
31
Polarity
- Recognises when section of mRNA x translated e.g. w/ ribosome
- regulator mechanisms that modulates expression
- Loss of ribosome allows rho-independent terminator to form over ORF
- OR loss of ribosome allows Rho to bind + termination to occur
- Rho-dependent termination site within a transcription unit usually masked
- Nonsense mutations release ribosome
32
Antitermination
- Used to control termination in phage + bacterial operon
- Antitermination = mod of E that allows it to read past a terminator into genes that lie ds
- Antitermination complex forms on Nut on RNA
- Nut sites have BoxA/B sequences where NusG + A assemble
- NusG = TF, +ve + -ve roles, CTD interacts w/ Rho
- NusA = NTD interacts w/ RNAP near RNA exit channel, SKK domain binds ssRNA of nascent transcript, nusA-SKK domain blocks rho-dependent termination
- NusA + G change properties of TEC via direct interactions (change processivity and slows RNAP)
33
Transcription attenuation
- Several operons controlled by termination in 5'UTR
- RNAP pausing synchronises position of RNAP w/ folding/regulatory binding
- B subtilis (protein-mediated or uncharged tRNA-mediated, when [Trp] ↓, TRAP x active, x bind RNA, RNAP overcomes pause, anti terminator readthrough into structural genes , when ↑ Trp, TRAP binds, prevents anti terminator forming, charged tRNA availability = sensed by operon, product at operon = AT, binds TRAP
- NusA-NusG pausing ↑ TRAP efficiency
34
Non coding RNA regulation
- Ribosensors = elements in 5' UTR of mRNA
- SAM riboswitch = us fo genes that code for proteins in Meth
- When SAM bound to anti terminator, anti terminator sequesters terminator so x form + Pol reads ds
- If SAM binds aptamer, terminator forms
35
Stringency
- Stress response in bacteria in response to aa/FA
- RelA factor makes alarming when starved
- pppGpp synthase = assoc w/ ribosome
- Targets of alarming = 7 rRNA operons, P1 = strong, us, P2 = strong, ds
- Other factors regulate rrn promoter, including DNA UP element, TF Is
- pppGpp traps stab closed/open configuration, slowing down elongation
- ppGpp accumulates affects resources, binds RNAP + changes transcription
36
Translational regulation
- Most important = interaction bte 3' end of 16S rRNA w/ SD/RBS us of initiating AUG in mRNA
- 3 ways: 2o structure in mRNA affect ends/exonuclease, protein that bind mRNA, RNA binding to mRNA
- Example = repression of translation by binding metabolite that binds alternative mRNA 2o structure, leaves SD in bp region e.g. Trp operon
- Example = repression of translation by formation of alternative mRNA 2o structure due to Δ temp
37
mRNA In bacterial translational regulation
- Translational inhibition by protein binding
- 30S subunit + Li protein compete for mRNA
- Preferentially bind rRNA
- Li binding mRNA → x ribosome binding so translation of rplk
- Entrapment mechanism, inhibits translation, traps 30S subunit during initiation e.g. protein 515
- Translation regulation by nc transcript
- Gene silencing by natural antisense RNA in bacteria
- Trans-antisense encoded w/ limited complimenarity to target mRNA
- Once antisense RNA bound, translation of target gene silenced
- small RNAs interact w/ rpos transcript to activate transcription initiation through interaction w/ mRNA 2o structure
