Eukaryotic Gene Regulation As in bacteria much gene regulation takes place at the level of transcription, although there are more examples of regulation at other levels. Some differences - no anti-termination or regulation of elongation as in prokaryotes Eukaryotes can regulate splicing and transport Eukaryotes have much more complex genomes Chromatin effects Enhancers How to study transcription? transcription assays in vitro transcription systems in vivo site directed mutagenesis introduction into heterologous systems binding studies in vitro gel retardation footprinting in vivo nuclease hypersensitive sites in vivo footprints fluorescent imaging sequence comparisons (TATA box) genetics promoter bashing fusion proteins transgenics knockouts What regulates transcription? chromatin effects euchromatin or heterochromatin? Euchromatin has higher transcription methylation state of the DNA? Less methylated regions show higher transcription locus control regions in a loop? Genes in loops have more transcription cis regulatory sequences and trans acting factors promoter sequences (different for each polymerase) Pol I - a core and an upstream control element Pol II - TATA (-25 &endash;-40) box and upstream elements Pol III - internal A and B boxes enhancer sequences (Pol II only) orientation and position independent effects Can be 5' or 3' of the gene Can be flipped backwards ant still work Can be thousands of base pairs away can also repress transcription (silencers) one gene may have many different enhancers each enhancer is responsible for expression in a particular tissue or at a specific time boundary elements - block enhancers from other genes Transcription factors a DNA binding domain helix-turn-helix - cro, l repressor, CAP, homeobox zinc finger - TFIIIA (9), SP1, steroid hormone receptor leucine zipper - fos, jun helix-loop-helix - c-myc copper fists - ACE1 (yeast), metallothionine promoter basic domain - CAAT binding protein an activation domain acidic - Gal4 glutamine-rich - Sp1 (39/143), Oct1 Proline-rich - CTF (19/84) Basic model is that chromatin effects determine whether a gene is 'competent' to be expressed and then cooperative interactions between proteins bound to the enchancer and to the proximal promoter determine whether the gene will be expressed Post-transcriptional regulation splicing (Sxl) degradation pulse-chase experiments can be used to measure the half-life of a mRNA casein mRNA half&endash;life increases from 1.1 hours to 28.5 hours after prolactin treatment translational control (usually effects large numbers of messages) in early development heat shock post&endash;translational proteolytic clipping removal of signal peptide processing (proinsulin) modification phosphorylation methylation glycosylation transport stability allosteric try answering these questions about eukaryotic gene expression from the Biology Project: question1, question2 [ Biol 207 ] [ Bell ] [ CSU Chico ] [ Library ] This document is maintained by: Jeff Bell Last Update: Wednesday, April 12, 2000