Adenovirus Promoter Experiment

Promoters in E. coli were found using bacteriophages like λ and T7, which act very strongly to encode massive quantities of viral proteins. This same idea was expanded to eukaryotes, using adenoviruses (which infect eukaryotic cells). Viral genes express massive amount of proteins and a great model for finding examples of eukaryotic promoter sequences.

adenovirus promoter experiment diagram

Nuclear Run-On	A modified in-vitro nuclear run-on experiment was performed on cells that had been infected with an adenovirus; the hybridization step is below. Viral genes express massive amount of proteins and a great model for finding examples of eukaryotic promoter sequences.
Centrifugation	The RNAs were separated by size using rate-zonal centrifugation. These adenoviral RNAs were added to a tube containing a gradient of sucrose with ↓ at the top and ↑ at the bottom. Centrifugation allowed the RNAs to be separated by weight (and, accordingly, by length).
Hybridization	The RNA segments were hybridized to endonuclease-digested adenovirus DNA. Logically, longer RNA bound more DNA segments, while shorter RNA segments bound less. A loose adenovirus genome map was made by recording which DNA segments were bound by RNA segments of increasing length.
Filtration	Hybridized DNA was obviously from coding regions and remained bound to the filter. Any ssDNA was thus a putative control region, and was eliminated from the filter. Unhybridized RNA was eliminated by RNase A digestion (which targets ssRNA).
Repetition	At various stages of the adenovirus life cycle, if a certain DNA segment never binds to RNA then it must not encode any RNA. If they are not coding regions, then these DNA segments must be control regions instead. A single adenovirus promoter was found for all late phase genes.
Synthesis	Regulation of TF synthesis (transcription of the TF gene).
Activity	Regulation of transcription factor (TF) activity (activators and repressors). Regulation of TF activity by interaction with small molecules (ligands), and post-translational modifications, especially phosphorylation and dephosphorylation. Regulates Nuclear transport, import and export; DNA binding to cognate DNA site; Interactions with co-activators.
Degradation	Regulation of TF degradation.