Chapter 6 Flashcards
What are the components of an E. coli polymerase holoenzyme?
β’, β, σ, α (2x) , ω
T/F: The ω component is necessary for holoenzyme function.
False
T/F: SDS-PAGE gels denature.
True
Core refers to:
The holoenzyme without the σ subunit.
T/F: Transcription is less efficient when σ is missing from core.
True
What would you expect to see transcription-wise if you had nicked eukaryotic DNA with holoenzyme?
Normal transcription
What would you expect to see transcription-wise if you had nicked eukaryotic DNA with only core?
Medium transcription
What would you expect to see transcription-wise if you had intact bacteriophage DNA with holoenzyme?
Normal transcription
What would you expect to see transcription-wise if you had intact bacteriophage DNA with only core?
Only basal transcription
What is necessary for RNA polymerase to have specificity?
σ is required, otherwise both strands will be transcribed
What is specificity?
Action where only the template strand is transcribed and the coding strand is not
Describe the experiment used to show specificity in transcription.
DNA was incubated with core and holoenzymes. RNase was added to each. RNase only works on single-stranded RNA, so if transcription was non-specific, the RNase would not work to degrade the molecules. ~30% was found to be resistant to RNase.
Define promoter
Polymerase binding site.
What can a filter binding assay determine?
Amount of protein-DNA binding. Temperature at which there is more binding affinity. How binding affinity is affected by salt concentration.
Describe the process and theory of a filter binding assay.
Double-stranded DNA will not bind to a nitrocellulose filter, while single-stranded DNA and proteins will do so. dsDNA can be combined with proteins and then run through the filter to determine the strength of binding because the dsDNA-protein complex will bind. Measurement of the quantity of DNA on the filter is then measured (often radio-labeled DNA which is then measured with a scintillator).
Specifically, the two can be mixed together and aliquots taken at intervals. From this a curve can be obtained to show binding over time.
What are the weights of the components of the holoenzyme?
β - 160 kD
β’ - 150 kD
σ - 70 kD
α - 40kD
ω - 10 kD
Which of the following are true about σ?
a. σ directs RNA polymerase to start at specific promoters.
b. σ is not necessary for transcription to occur in a specific manner.
c. σ can be recycled.
d. Two of the above.
e. None of the above
d (a and c)
What are the steps in performing a filter binding assay?
- Purify enzymes.
- Incubate with 3H labeled DNA whose promoters are recognized by the polymerase being used.
- Add excess unlabelled DNA.
- Over time, remove aliquots and pass them through a nitrocellulose filter.
- Measure radioactivity on the filter with a scintillation counter.
In the filter binding assay, the level of holoenzyme bound to DNA remained fairly constant over time, while the amount of core bound to DNA diminished. Interpret these results.
Holoenzyme binds more tightly to DNA than core. Becuase the level stayed fairly constant, it was not dissociating from the DNA and then being outcompeted by the excess DNA. With core, the amounts quickly decreased because as core fell off, it was being outcompeted by non-labelled DNA.
START AT PAGE 3 OF NOTES
FOLLOWING IS LECTURE 2, IN-CLASS
Does σ always dissociate from core after initiation?
No, it may be random. Alternatively, experimental methods may have caused σ dissociation, so it may not happen at all in vivo.
What does FRET stand for?
Fluorescence Resonance Energy Transfer