EXAM 3 REVIEW

Trp review:
- Ribosome sitting on trp attenuator
o Mutations in attenuator
- Aporepressor = not active repressor
o When made = no activity
o Trp must bind  becomes active
o Trp is the end product of the pathway
o Trp = corepressor
o Aporepressor + trp => binds operator – and shuts down activity – responsible for
70-fold of regulation (negative regulation)
o Also used to regulate its OWN promoter (repressor operon – not part of the
proper operon)
o Sits down and gets in the way of binding RNA polymerase
- Attenuation = gets all the press
o Only responsible for about 10% of regulation
o Is there sufficient trp?
o trpL = there for regulation – stop of transcript possible = attenuator
 testing to see if the pol has to pause and see if there’s enough trp
o this is where the 1,2,3,4 hairpin partner thing is
o 1&2, 2&3, 3&4
 3,4 forms hairpin = transcription terminates, string of Us and As on
template strand
 1,2 & 2,3 = pause but no termination
 If you can prevent 3&4 together = get no termination, can tie 3 up
(hairpin up with 2) = how control 2’s ability to attract 3? Ribosome!
 If ribosome stops at TrpTrp (at 1) then 2&3 will bind up
 Binary switch –
 stops early (at TrpTrp), trp shortage (want 3&4 not to happen) =
2&3 bind  transcription happens
 Plenty of trp, ribosome will go all the way to the stop codon in
region 1, so 2 is blocked, therefore 3&4 will bind = termination will
happen
 Anti-termination is the 2,3 hairpin
 3,4 is the intrinsic terminator
 Think – too much trp? want termination to happen = terminator happens,
make sure 2 can’t interfere, 2 is tied up, ribosome blocking it; 3&4 hairpin
=> termination
 HE WILL PUT MUTATIONS there:
 No TrpTrp in region 1, would always go through, no reason to
pause, 2 will always be blocked, always terminate – never ever be
able to make trp
 Anti-terminator = enemy of shutting down
 - If you have rainbow pairing (hairpin), then AAAAA in active site; falls apart – termination

CAP
- Class I, II, III
- Cap = purely for activation, gate keeper for catabolic repression, positive activating,
there to enforce glucoses domination; glucose in media someplace, E. coli LOVES
glucose, designed to enforce bias; make all other pathways on the positive activing
transcription factor; -35 is almost non-existent; CAP binds upstream, makes contact with
pol, increases affinity for pol to the promoter
- CAP will not bind unless it has found a small signal molecule (cAMP) – dependent
- Lac – IPTG present, no longer fit into the operator – only one monomer of dimer can
bind
o CAP opposite – when binds cAMP, when they bind, dimer moves to fit perfectly
into region its binding, simultaneously
- Binds as a dimer, creates kink
- Slave to glucose
- Glucose there, no CAP; CAP there, no glucose
Sugars
- Lac and ara are glucose dependent
- Is there inducing substrate there (lactose, arabinose?) – is there a dna loop?
o Loop = bad news; loop yes, sugar no

Pic of lac/ara operon,  be able to say if glucose is there and/or specific sugar is there
Gene on or off?
- NO CAP MEANS gene is OFF
- No CAP = no go!
- Loop = no go
- LacR bound (not to operator though) = lactose is there, inactivated the repressor (no
loop too)
o If LacR was bound to operator – no lactose (would be looped)
- Lac operon = negative control & positive control
Arabinose
- Loop = shuts down = gets in the way of RNA pol, can’t’ have access to promoter
o Loop (in lac) = binding for pol is on either side = provides torsional resistance;
sigma has a hard time melting, must bind 2 places simultaneously;
 Glucose +, CAP –, lacR+ (at operator); IPTG arrives, IPTG binds between
the two cores (separate, no longer fit on operator); helix 4 is going to
come out of minor groove, one of dimers can’t fit anymore, loop springs
open, the one part of the LacR has increased the RNA pol affinity for the
promoter, IF THERE’S NO CAP THOUGH its not active
- araC mimics CAP and binds at -41..5 when arabinose comes, provides high affinity site
for RNA pol;  class III – needs 2 activators (one is CAP, other is usually araC)
GALACTOSE PROMOTER
 - Class II promoter; sitting at -41.5 only; facilitates melting sigma; affects transition from
CLOSE TO OPEN complex
- List of promoters, looking at names, could you tell me which ones have a good -35 and
those that don’t?
o Anyone that is CAP dependent has lousy -35, lac, ara, gal (all use CAP); any
biosynthetic pathways have good ones;
Basis
REPAIR
- Figure has mutS,L,H drawn, pale blue background; MIGHT SEE on exam, arrows labeling
o Show me protein that connects to B camp – not straight forward
o Know what each one doe
o H – endonuc. that can’t bump unless L bumps, must determine which strand to
cut, looking for unmethylated strand to cut;
o L – used to have nuclease activity; nothing has H except E.coli and other ..
o S recognizes – IDs mismatch; binds and changes config; L comes; travel away
from mismatch; must find which strand to cut; bump into H; L is connected to H
and S; cut made in new strand; peel back all the way to mismatch; uvrD comes in
to unwind; now use pol III to synthesize the strand (w/ PCNA(euk)/B
clamp(bacteria))
o L has affinity for everything – for S H uvrD B-clamp/PCNA
Mut T, M, Y
- Base modification
- T = nucleotide
- M = marriage – two become one
- Y = adenyl glycoseylase
- Slippage = MutS Homolog (MSH) 2/3
- Single mismatch = MSH 2/6
- Rav4 = XPC = job: recognizes mismatch in global scanning
- Uvr A,B,C,D
o AB – recognizes (or Mfd recognizes and recruits if UvrAB isn’t there already)
o A leaves, ATP required
o B calls C , C cuts ; ATP required
o D unwinds (ATP)
o Pol 1 fills in gap (day job: repairing dna replication in lagging strand)
Deamination products
- CU
- 5-Methyl-C  T
LexA
- Error prone repair
- LexA system represses high activity that cause error prone to happen
o Represses ITSELF too
- It can be regulated because it can cut itself in two
- Auto-proteolysis (must be induced by RecA)
 - RecA causes it to cut itself in two
- UV activates RecA (other target, subunit of error prone dna pol (pol 5- umuD)
- Dual promoters - allows for rapid turn off – once out of mutagenesis woods, want to
shut it all down and put it back in its cage
HW questions
- Temp effect on promoter
- Footprinting
 No questions on this stuff
Retrieval Repair
- Thymine dimer – stops dna replication – transcription stopped
- Doesn’t know how to put As across from two Ts
- Must figure out to get rid of Ts and continue
- 1st must figure out what’s across, want to repair Ts when you know what s opposite of
them; must find original other strand and retrieve it
- Involves recombination  RecA involved in this kind of repair
- RecF and RecG involved too
- Goes forward, backs up, etc. Holiday structure happens

Ape1
- Recruits replicases pol d/e (long-patch) or pol b(short patch)
o Ape1 makes decision – enzyme that introduces nick
 If Ape1 makes nick itself, recruits d/e but if other enzyme nicks it, recruits
b
o Glycosylase first removes base
o Nick must either be Ape1 or recruits something else for it
o Look at diagram in slides
TFIIH
- XPB & XPD
- Rad3 = XPD  3D  recognize
Functional domains of sigma
- Label one that pokes its way through exit pore == 3.2
- 3.1 lays in channel before dna gets there
- 2.3 melts
- 2.4 recognizes TA of -10
- 3 recognizes extended TATA
- Discriminator?
- -35 contact = 4.2 domain
- Interact with minor groove = alpha-CTD (hairpin helix) and hinge helix (of lac repressor =
helix 4)
- Leucine zipper purpose = binds the two dimers together to make tetramer
- Sigma & core ratio 1:3 (pie chart); 50% core, 25% sigma, 25% elongation
- Triggering event causes transition from initiation to elongation?  sigma bumped in
face, nascent transcript finally pushes it out, sigma lets go,
- DNA scrunching! – transcribe abortive transcripts
- Most conserved domain – domain 2 generally – where it makes contact with the core
- Sigma 54 – what other protein does it need to be fully function – nTRC?
o Allows it to finally melt a promoter  can’t melt promoter on its own (because it
isn’t locked up with core)
o Missing 1.1 domain
 Allows it to act like euk. transcription factor
 Allows it to bind on its own
 Its not bent back masking 4.2
 Therefore doesn’t need to bind to core to unbend/unmask it
- If sigma54 doesn’t have to bind core first – always downstream of promoter that uses
sigma70 (constitutive one); opportunity to see CORE, sigma is almost acting like a
repressor; gets close to Core, little switcharoo going on there? Maybe they switch
sigmas? Sigma54 maybe replaces regular sigma (70)  professors idea of how it
happens?