molecular exam 2 - bacterial transcription & operons

basic transcription

• transcribe DNA → RNA

• need:

  • RNA polymerase: enzyme that recognizes DNA at promoter, forms open promoter complex, uses template strand to synthesize DNA from 5’ → 3"‘ ends

  • DNA template

  • rNTPs: ribonucleotide triphosphates

• initiation, elongation, termination

components of bacterial RNA polymerase

• RNA polymerase = holoenzyme complex

  • holoenzyme complex = core + σ factor

  • core made up of:

    • α peptide

    • β peptide

    • β’ peptide

• σ factor is loosely associated w/ core

necessity of σ factor

• holoenzyme (σ factor + core):

  • σ factor allows specific & strong interactions with promoter seq.

  • once σ factor dissociates from the complex during elongation, core transcribes seq.

• core (w/o σ factor):

  • does not interact w/ promoter, transcribes nonspecific RNA

• σ factor greatly enhances the binding capability of core to DNA

• can see this via filter binding assay:

  • nitrocellulose filter

  • DNA runs thru, while protein is retained

  • if DNA is bound to protein, it gets stuck on filter

  • can label DNA & detect radioactivity/fluorescence on filter to detect binding capability

promoter structure

• holoenzyme complex recognizes particular target seq. to start initiation

• -10 box: TATA box - upstream from initiation start site

  • recognized by region 2.4 on σ factor

• -35 box

  • recognized by region 4.2 on σ factor

• UP element: upstream promoter element, consensus sequence, close to -35 box

  • recognized by α peptide

• when there is a closer match to the consensus seq. = stronger expression

• weak match to consensus = more regulated expression

dissociation of σ factor

• question: does dissociation happen a lot? is core dead once σ factor dissociates? can σ factor be reused?

• experiment mechanism:

  • holoenzyme + DNA template = transcription rxn

  • once holoenzyme finishes elongation phase, it wants to dissociate, but bc of low ionic conditions, it can’t & remains associated w/ seq.

  • after, add purified core (no σ factor) & see huge inc. in transcription

  • conclusion: σ factor originally present in 1st holoenzyme complex dissociates & re-associates w/ new core for continuation of transcription

timeline of σ factor dissociation

• FRET experiments to determine when σ factor dissociates from core during transcription

• mechanism:

  • trailing-edge FRET: doesn’t work bc fluoro added on trailing edge of DNA template & σ factor

    • detects movement of σ factor on strand bc of dec. in fluoro detection

    • problem: don’t know if dec. in fluoro is due to dissociation of σ or just bc complex is advancing on seq.

  • leading-edge FRET: fluoro on leading edge & σ factor & look for emergence of fluoro

    • if σ is released from core, get dec. in fluoro

    • if σ is retained on core, get inc. in fluoro

• see that most core lose σ factor around elongation

• indication that DNA sequence influences if σ is released

abortive transcription

• since σ factor causes strong association of the holoenzyme complex & DNA template, it takes a few tries to move on from the promoter to go into elongation phase

  • can see this by using radioactive rNTPs: get many short RNA sequences produced before complex is able to enter elongation phase

• mechanisms (3 options): do FRET experiments to figure out which one is right

  • transient excursion: RNApol repeatedly moves forward, then comes back

    • w/ trailing edge FRET, promoter on upstream part of DNA: would expect FRET signal to dec. bc have movement away from promoter

    • ruled out bc see no change in FRET

  • inchworming: RNApol repeatedly stretches itself out

    • w/ trailing edge FRET, would expect no change in FRET bc RNApol is not moving positions on back end

    • w/ leading edge FRET, promoter on downstream part of σ: woould expect dec. in FRET bc RNApol is elongating & causing increased separation of fluoros

    • ruled out bc see no change in FRET

  • scrunching: repeatedly pulling DNA into itself & releasing it

    • w/ trailing edge FRET, would expect no change in FRET bc RNApol is not moving positions

    • w/ leading edge FRET, would expect no change in FRET bc length of σ is not changing, moving DNA instead

    • w/ fluoro on promoter & downstream DNA: would expect change in FRET

      • see this!

• scrunching is mechanism of clearance: RNApol pulls in ~6-9 NTs, but once transcript becomes ~10 NT long, it is enough for clearance

β’ interactions w/ σ

• σ factor doesn’t bind DNA well on its own, requires core for effective binding

• evidence that β’ interacts w/ σ

• mechanism:

  • mix β’ & σ & use single stranded radioactively labeled DNA

  • β’, σ, & DNA form complex, & when hit w/ UV light - causes covalent bond

  • SDS-PAGE to see if σ is associated w/ the DNA

  • can take smaller & smaller regions of β’ to see what region of β’ is required for σ factor binding

    • see that β’ fragment 262-309 is sufficient to promote σ-DNA binding

lac operon info

• bacteria prefer to use glucose as main carbon source, but if glc is not present, can activate lac operon to be able to use lactose

• components:

  • lacI: shows constitutive expression, produces repressor monomers that are being made at low levels all the time

    • monomers assemble into tetramer: lac repressor, binds to operator sequence & when bound, it inhibits/blocks transcription

    • when lactose is present, allolactose is present: inducer molecule, binds to tetramer & inhibits binding, allowing for RNApol II to bind to promoter sequence & transcription of downstream elements:

  • lacZ: codes for β-galactosidase - enzyme that breaks down lactose

  • lacY: codes for lac permease - transporter protein that allows for cells to take up lactose from their environment

  • lacA: codes for lac transacetylase - acetylate galactosites, especially ones that are potentially toxic to be cleared & removed from cells

  • lac promoter: where σ factor recognizes (-10, -35 box) for binding of RNApol II

    • has deviations from consensus seq. - very weak promoter

mutations of lac operon

• mutant repressor gene: I^-

  • causes no production of repressor, so lac operon is always on, even in absence of lactose

  • diploid: WT + I^- : WT allele masks effects, repressor made by WT is able to repress mutated operator also - mutation is recessive to WT

• mutant operator: O^C

  • operator no longer binds repressor, causes constitutive expression

  • diploid: WT + O^C : mutation is cis-dominant (mutation only effects sequence it is on, WT sequence functions normally)

• mutant repressor gene: I^S

  • produces repressor that can’t recognize the inducer - represses under all conditions

  • diploid: mutated repressor & WT repressor form complex that is unable to bind allolactose - mutation is cis & trans-dominant (mutation effects both the WT seq. & the seq. it is on)

• mutant repressor gene: I^-d

  • produces repressor that can’t bind the operator seq. - causes constitutive expression

  • diploid: mutated repressor & WT repressor form complex that is unable to bind operator - mutation is dominant-negative (mutation causes constitutive expression in both sequences)

3 lac operators

• sequences can contain 3 lac operators:

  • O1: primary operator, when it is lost - big drop off of strength of repression

  • O2

  • O3

• all 3 operators are required for optimum repression

  • when one or multiple are lost, see significant drop in the ability of the repressor protein to repress transcription

• lac repressor is capable of repressing promoters that lave all 3 operator sequences

• repressor protein is a tetramer of 2 dimers that contain their own binding domains

  • one dimer binds one operator sequence, while the other binds to another

  • since the dimers are connected: creates looping activity of the sequence - further restricts access to the promoter

cAMP + CAP activity

• CAP + cAMP helps RNApol bind to the promoter & establish the open promoter complex

• during glucose starvation (& ∴ bac has to switch to lactose utilization), high levels of cAMP produced

• CAP + cAMP form a complex w/ CAP binding site very close upstream to where RNApol binds the promoter

• when CAP+cAMP binds DNA, forms kink, & connects w/ the c-terminal domain of the α subunit of RNApol

• not all promoters have a CAP binding site, so only promoters that have this site have increased transcription when CAP+cAMP is present