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final_Yin - Summary(methods-DNA RNA or protein can be...

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Summary (methods) -DNA, RNA or protein can be detected by Southern, Northern or Western blotting ; microarrays can be used to detect the expression of almost all the genes in the genome -Several experiments are widely used for DNA:protein interaction studies GMSA : test in vitro binding of proteins to DNA Footprinting : map the DNA binding sites of a protein in vitro ChIP : confirm the DNA:protein interactions in vivo Chip-on-chip : identify all the target sites for a transcription factors in vivo -Methods for protein:protein interactions GST pull-down: confirm direct interaction in vitro Yeast two-hybrid : test protein interactions or identify protein partners Immunoprecipitation: confirm protein:protein interactions in vivo
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Regulation of RNA Polymerase II preinitiation TATA box enhancer Pol II F H E DB AC DNA binding Activation Transcription activator Mediators and Coactivators TFIID (TBP+TAFs) A TFIIB
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Summary: Eukaryotic promoters Class II promoters have TATA box, Initiator (Inr) and upstream elements. TATA box determines the transcription start site (about 30 bp downstream) Upstream elements, regulating the rate of transcription, can be identified by making promoter mutations and assaying the promoter activity
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Function of TFIID TBP (TATA box-binding protein) -binds TATA box at the minor groove and bends DNA to start transcription initiation. TAF II s (TBP associated factors) -recruit TBP to TATA-less promoter to start transcription initiation (TAF II 250 and TAF II 150 bind to initiator and DPE, TAF II 250 and TAF II 110 interact with Sp1 that binds to GC box) -different TAF II s are required to respond to various activators - TAF II 250 has histone acetyltransferase (HAT) and kinase activities that modify chromatin and other transcription factors
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Function of TFIIB TFIIB serves as a linker between TFIID and RNA Pol II/TFIIF -the N-terminal domain interacts with Pol II, the DNA and RNA in the active center -the C-terminal domain interacts with Pol II, TBP and DNA, and orients the DNA for unwinding and transcription.
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Function of TFIIF, TFIIH and TFIIE -TFIIF binds Pol II and interacts with non-template DNA strand -the TFIIH phosphorylates CTD of Pol II, which may be required for transition from initiation to elongation -TFIIE stimulates CTD phosphorylation by TFIIH -TFIIH has a DNA helicase activity that is essential for transcription, because it causes full melting of the DNA at the promoter.
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Transcription Activators in Eukaryotes: DNA-binding domains 1. Zinc-containing modules a. Zinc fingers (2His, 2Cys and 1 Zinc) b. Zinc modules in nuclear receptors (8 Cys and 2 Zinc)
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