BCH5413 TEST 2 QUESTIONS AND ANSWERS ALL
REVISED AND UPDATED
Stages of transcription initiation - ANSWER -- holoenzyme recognizes
regulatory sequence in promoter and binds forming the closed promoter
complex
- unwind dsDNA to form open promoter complex
- transcription initiates (RNA Pol generates short abortive transcripts
that are unstable and released)
- at 10nt in length, the complex is stable and turns from initiation to
elongation
Cycling of sigma during transcription - ANSWER -- as Pol transitions
to elongation, sigma factor is released
- transcription proceeds with just core subunits until termination
- at termination, RNA Pol released
- nascent RNA released
- released core enzyme recycles back to bind with sigma to form the
holoenzyme and repeats
Abortive transcription and initiation: RNA Pol at promoter - ANSWER
-3 ideas:
1. movement of transient excursion: RNA Pol makes short movement
along DNA to make abortive transcripts, then goes back to its original
position
2. inch worming: back of RNA Pol stays stationary, as abortive
transcripts are made, front edge of RNA Pol stretches/inch worms, as
-
,abortive transcript is released front of RNA Pol goes back to initial
position
,3. scrunching: RNA Pol doesnt move but DNA compresses/scrunches
in promoter
How scientists determined which theory about RNA Pol and abortive
transcription is true - ANSWER -FRET
- donor and acceptor used; donor can be stimulated and release energy
which is transferred to acceptor and will fluoresce at different
wavelength
- efficiency determined by proximity between donor and acceptor
How FRET would work concerning RNA Pol and abortive transcription
- ANSWER -- linked acceptor to DNA template and donor to back end
of sigma subunit
- assembled open promoter complex
- GTP and UTP added to start synthesis
- measured degree of energy transfer between donor and acceptor
(proximity)
What FRET would show for:
- transient excursion
- inchworming
- scrunching - ANSWER -- transient excursion: as Pol moves forward,
FRET will drop
- inchworming: no change (no net separation)
- scrunching: no change (no net separation)
What FRET showed - ANSWER -- no change; eliminated transient
excursion model
, How to distinguish between scrunching and inchworming
- what inchworming would show
- results - ANSWER -- acceptor on DNA and donor at front of sigma
subunit
*if inchworming, FRET would decrease
results: no change in FRET
How they directly determined scrunching was the correct model
- what FRET would show
- results - ANSWER -link donor and acceptor on DNA
- if scrunching occurs, FRET would increase
results: showed increase in FRET
What is scrunching required for? - ANSWER -promoter clearance
Initiation complex
- RNA Pol initially contacts the region from....
- when sigma dissociates, the core enzyme contracts to...
- when the enzyme moves a few base pairs.... - ANSWER -- RNA Pol
initially contacts the region from -55 to +20
- when sigma dissociates, the core enzyme contracts to -30 to +20 -
when the enzyme moves a few base pairs, it becomes more compactly
organized into the general elongation complex
Elongation
- rate of elongation with E. coli RNA Pol - E. coli RNA pol covers..
REVISED AND UPDATED
Stages of transcription initiation - ANSWER -- holoenzyme recognizes
regulatory sequence in promoter and binds forming the closed promoter
complex
- unwind dsDNA to form open promoter complex
- transcription initiates (RNA Pol generates short abortive transcripts
that are unstable and released)
- at 10nt in length, the complex is stable and turns from initiation to
elongation
Cycling of sigma during transcription - ANSWER -- as Pol transitions
to elongation, sigma factor is released
- transcription proceeds with just core subunits until termination
- at termination, RNA Pol released
- nascent RNA released
- released core enzyme recycles back to bind with sigma to form the
holoenzyme and repeats
Abortive transcription and initiation: RNA Pol at promoter - ANSWER
-3 ideas:
1. movement of transient excursion: RNA Pol makes short movement
along DNA to make abortive transcripts, then goes back to its original
position
2. inch worming: back of RNA Pol stays stationary, as abortive
transcripts are made, front edge of RNA Pol stretches/inch worms, as
-
,abortive transcript is released front of RNA Pol goes back to initial
position
,3. scrunching: RNA Pol doesnt move but DNA compresses/scrunches
in promoter
How scientists determined which theory about RNA Pol and abortive
transcription is true - ANSWER -FRET
- donor and acceptor used; donor can be stimulated and release energy
which is transferred to acceptor and will fluoresce at different
wavelength
- efficiency determined by proximity between donor and acceptor
How FRET would work concerning RNA Pol and abortive transcription
- ANSWER -- linked acceptor to DNA template and donor to back end
of sigma subunit
- assembled open promoter complex
- GTP and UTP added to start synthesis
- measured degree of energy transfer between donor and acceptor
(proximity)
What FRET would show for:
- transient excursion
- inchworming
- scrunching - ANSWER -- transient excursion: as Pol moves forward,
FRET will drop
- inchworming: no change (no net separation)
- scrunching: no change (no net separation)
What FRET showed - ANSWER -- no change; eliminated transient
excursion model
, How to distinguish between scrunching and inchworming
- what inchworming would show
- results - ANSWER -- acceptor on DNA and donor at front of sigma
subunit
*if inchworming, FRET would decrease
results: no change in FRET
How they directly determined scrunching was the correct model
- what FRET would show
- results - ANSWER -link donor and acceptor on DNA
- if scrunching occurs, FRET would increase
results: showed increase in FRET
What is scrunching required for? - ANSWER -promoter clearance
Initiation complex
- RNA Pol initially contacts the region from....
- when sigma dissociates, the core enzyme contracts to...
- when the enzyme moves a few base pairs.... - ANSWER -- RNA Pol
initially contacts the region from -55 to +20
- when sigma dissociates, the core enzyme contracts to -30 to +20 -
when the enzyme moves a few base pairs, it becomes more compactly
organized into the general elongation complex
Elongation
- rate of elongation with E. coli RNA Pol - E. coli RNA pol covers..
