Mechanism and Control of Gene Transcription I

Question 1

Regulators of transcription

Answer 1

1. TF
   - Regulate expression of genes +vely and -vely
   - Act primarily to control transcription initiation
   - E.g. = sigma factors, activators
2. Nc- RNA
   - Regulate expression of genes +vely/-vely
   - E.g. = ribosensors/switches
3. DNA topology
   - Signals for regulation are often environmental

Question 2

DNA topology

Answer 2

1. 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
2. Nucleoid
   IHF bends DNA, bacterial basal expression occurs throughout the nucleiod, loop-like structure helps Pol find promoter
3. Toposiomerase
   Bacterial chromosome = -vely supercoiled, +ve supercoils relax DNA, changes either improve or worsen

Question 3

How RNAP finds promoter

Answer 3

• Promoter search → conformational change from closed to open → abortive initiation → promoter escape

Question 4

RNA pol structure

Answer 4

• a = regulatory subunit
• B and B’ form active site, similar to RBP1 + 2 in eukaryotes
• σ = in holoenzyme, needed for initiation
• Also have w

Question 5

Sigma factors

Answer 5

• Different promoters have different factors
• 2 main classes: σ54 + σ70
• σ70 = binding at -10, -35
• σ54 binds at -24, -12

Question 6

RNA pol binding to promoter

Answer 6

• 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

Question 7

Conformational change

Answer 7

• 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

Question 8

Other factors

Answer 8

1. ECF σ factor
   Part of σ70, -10 specific, modulate response to environmental condition, most co-transcribed w/ operons
2. Anti-signa factor
   ASF = TM protein that binds to + inhibits cognate σ factor

Question 9

σ70 vs σ54

Answer 9

• 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

Question 10

σ54 melting

Answer 10

• x melt DNA unlike σ70 (no K2)
• In initial inhibited state, σ54 blocks template DNA from entering RNAP AS
• Enhancer binding proteins

Question 11

bEBP

Answer 11

• 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

Question 12

σ70 Activators (CRP/CAP)

Answer 12

• 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

Question 13

σ70 activator

Experiment

Answer 13

1. 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
2. Mutate DNA in operon + screen bacteria that induce operon in presence of glucose + induce, lacUV5 promoter

Question 14

FNR

Answer 14

• 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

Question 15

Regulating transcription

Repressors

Answer 15

• 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-

Question 16

Lac repressor structure

Answer 16

• 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

Question 17

Repression in lac operon

Answer 17

• 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

Question 18

Experiment J+ M

Answer 18

1. Jacob + Monod early
   - Lac- mutants isolate
   - 3 different substrates
2. 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

Question 19

Principles from J+M experiment

Answer 19

• 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

Question 20

Experiment

LacI- vs LacO-

Answer 20

• 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

Question 21

Initiation to elongation

Answer 21

• 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

Question 22

Abortive initiation

Answer 22

• 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

Question 23

Elongation mechanism

Answer 23

• 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

Question 24

Backtracking

Answer 24

• 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

Question 25

TF for backtrack recovery

Answer 25

• GreA + B are +ve elongation factors
• Mitigate pausing → reactivating arrested complexes
• Stimulate intrinsic hydrolytic cleavage of polymerase
• Enter 2nd channel of RNAP

Question 26

Backtracking mechanism

Answer 26

• 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

Question 27

Termination

Answer 27

1. Rho-independent (intrinsic)
   - Identified at sequence level
   - Step loop followed by U-rich sequence
2. Rho dependent
   - Requires Rho factor, distributed over genome

Question 28

Transcription elongation complex

Answer 28

• 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

Question 29

Intrinsic terminator

Answer 29

• 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

Question 30

Rho factor

Answer 30

• 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

Question 31

Polarity

Answer 31

• 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

Question 32

Antitermination

Answer 32

• 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)

Question 33

Transcription attenuation

Answer 33

• 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

Question 34

Non coding RNA regulation

Answer 34

• 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

Question 35

Stringency

Answer 35

• 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

Question 36

Translational regulation

Answer 36

• 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

Question 37

mRNA In bacterial translational regulation

Answer 37

• 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