22 - Transcription (Eukaryotes); RNAPII Promoters & Activators, Repressors

22 - Transcription (Eukaryotes); RNAPII Promoters & Activators, Repressors

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Eukaryotic regulation of transcription initiation Measurements of transcription rates reveal that regulation at level of initiation of transcription is widespread role of promoters and cis-acting elements In most cases (but not always): in transcription rate in cytoplasmatic mRNA in the corresponding protein Keep in mind: northern, in situ hybridization, microarrays information about steady-state transcript levels (mRNA abundance); based on extracted ( previously transcribed, processed, transported into cytoplasm and possibly already partially degraded ) RNA Cannot distinguish between transcription rate and degradation rate of mRNA
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Transcription rate in eukaryotes depends on: •availability/strength of the promoter , •presence/absence of regulatory proteins and •availability/strength of their binding sites (cis elements, boxes) on DNA How is the above different from Pro???? • Transcription rate(s) could be measured in vivo and in vitro : Run-off transcription check for accuracy of transcription rate of transcription Nuclear run-on transcription (nascent chain assay) Nuclei extracted and labeled with 32 P Relative transcription rates determined by measuring product radioactivity Reporter gene transcription and 5‟ deletion series of the promoter Reporter gene controlled by promoter of interest
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Run-on, run-off and reporter gene transcription (methods) mRNA is getting transcribed during the assay information about the actual efficiency of transcription – transcription rate These assays are used to locate AND assay the promoter region ( presence/absence of all necessary elements, their position and availability ) Start site of transcription Upstream cis regulatory elements located in this region (= promoter) Transcribed region Downstream cis regulatory elements may be located in this region Start site of translation Stop site of translation GENE
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Currently the most frequently used are reporter genes code for easily assayed gene products (usually enzymes): Idea : to investigate the strength of the promoter you clone the reporter gene after promoter of interest expression of the reporter gene is under control of promoter of interest level of expression of measurable (easy to assay) gene product (enzyme) is proportional to the strength of the promoter
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22 - Transcription (Eukaryotes); RNAPII Promoters & Activators, Repressors

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