Gene expression in prokaryotes and eukaryotes is tightly controlled, mostly via transcriptional control whereby the initiation of transcription is highly regulated. In prokaryotes, the promoter is where repressors and activators bind and it is usually near the gene it regulates. In eukaryotes, the promoter is relatively far away and is bound by transcription factors (the eukaryotes equivalent of repressors and activators). In higher eukaryotes, some transcription factors even bind at regulatory sites thousands of base pairs upstream or downstream from the promoter; and the same transcription factor can even bind multiple regulatory sites by bringing two sites close to each other (looping out DNA in the middle). In bacteria, control of gene expression is most often accomplished by regulating transcription initiation from promoters that are ~100 bp of the start site.
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.
| Step | Overview |
| 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. |
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| 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). |
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| 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. |
Another laboratory had been working out techniques to extract protein from nuclei so as to maximize in vitro transcription from isolated DNA (0.3 M KCl + buffers and reducing agents). They used this nuclear extract to perform in vitro transcription on restriction fragments of viral DNA from the region where the promoter was approximately mapped in the previous experiment.
Repressors are modular and have separate DNA-binding and repression domains. In repressor-directed histone deactylation, a histone directs the deacetylation of histone tails of chromatin. Activators are also modular and have separate DNA-binding and activation domains. They can direct hyper-acetylation in activator-directed hyperacetylation.
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