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Questions and Answers
What is the nature of sigma dissociation from RNA Polymerase?
At which position downstream of the promoter does experimental evidence show σ may be bound to the core?
What is the purpose of Fluorescence Resonance Energy Transfer (FRET) in testing the σ cycle hypothesis?
What occurs to the FRET efficiency when σ comes closer to the 3’end of DNA?
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What is the role of the probe on σ in FRET?
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What is the consequence of σ changing its association with the core during elongation?
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What is the effect of RNA polymerase on the DNA at the transcription start site?
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What is the characteristic of the bubble formed during transcriptional initiation?
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What is the consequence of local DNA melting at the promoter?
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What is the function of RNA polymerase during transcriptional initiation?
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Study Notes
Protein Domains and Proteolysis
- Limited digestion of protein domains reveals that they resist proteolysis, while unstructured regions between domains are susceptible to digestion.
- The α subunit has two domains connected by an unstructured linker of approximately 13 amino acids.
Model for Function of the C-terminus of α Subunit
- In a core promoter, the C-terminus of the α subunit is not required for function.
- With a UP element present, the C-terminus contacts the element.
Elongation and RNA Polymerase Subunits
- Core polymerase continues to elongate RNA after initiation and σ has left the holoenzyme.
- RNA polymerase subunits can be separated and reconstituted to re-create an active enzyme.
- Combining subunits from different sources can help understand their functions.
- Core polymerase determines sensitivity to rifampicin.
β Subunit Function
- The β subunit is responsible for sensitivity to rifampicin.
- The β subunit also determines sensitivity to streptolydigin, an antibiotic that blocks elongation.
- The β subunit is involved in phosphodiester bond formation during elongation.
UP Element Recognition
- The α subunit of RNA polymerase recognizes the UP element.
- The C-terminal portion of the α subunit is required for recognizing the UP element.
DNase Footprinting Assay
- The DNase footprinting assay shows that the α subunit contacts the UP element.
α Subunit Structure
- The N- and C-terminal domains of the α subunit fold independently and are tethered together by a flexible linker.
Sigma Dissociation
- Sigma dissociation from RNA polymerase is a stochastic event that may not always occur after initiation.
- Sigma may remain weakly bound to the core during elongation.
FRET Analysis
- Fluorescence resonance energy transfer (FRET) can measure the position of σ relative to DNA without separation techniques.
- FRET analysis shows that σ may change its association with the core during elongation.
Local DNA Melting
- RNA polymerase causes melting of 10-17 base pairs at the transcription start site, creating a "bubble" that moves with the polymerase.
- This melting exposes the template DNA.
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Description
This quiz covers the structure and function of the α subunit in molecular biology, including its domains, proteolysis, and interactions with promoter elements.
