BIOM 121 1st Edition Lecture 5 Outline of Current Lecture 1. Regulatory Elements2. Transcription factors3. Structure of Chromatin4. Post-transcriptional controlCurrent LecturePromoters – short (about 10 bp in length) located <200 bp upstream from start site. Docking sites for transcription factors. 2) Enhancers – 50- 200 bp in length. Can be 50,000bp upstream of start site, introns; and downstream. (GREEN BOXES) docking sites for transcription factors 3) Repressors – decrease transcription; (50-200 bp in length) found anywhere. Acts as a blocker so RNA poly doesn’t bind as well. House keeping gene- gene that is transcribed all the time at relatively low levels. Repressor would be used to downgrade the house keeping geneUse of a reporter gene to determine transcription-controlling regions – where upstream/ what elements are there Gene that makes an enzyme so we can visualize a color change Have luciferase (reporter gene) and insert gene Y upstream.Mutate or delete upstream areas, and use a reporter gene to determine transcription-controlling regions Transcription of reporter gene Measuring whether poly will transcribe reporter gene instead, by seeing if light shows.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.B. Transcription Factors- proteins that bind to DNA (promoters, enhancers, repressors) Binding of transcription factors recruits RNA poly to that site on the gene Without transcription factors bound to it, RNA poly will not bind Humans have about 2,000 TF’s. Often expressed in cell specific manner. (Lungs and Liver have diff TF’s) Also developmentally specific manner. (TF’s made as an infant is diff as an adult)Zinc finger motifs- fit into major groove of DNA; 2 alpha helixes fit Have interactions between TF to further ability to control gene expression. Have multiple TF’s that bind upstream from gene to mediate transcription Interactions between TF’s to more closely regulate transcription 1. Chromatin immunoprecipitation (ChIP) Treat living cell with formaldehyde Cross link a TF to the gene Break the cell open and have access to DNA, then use nuclease to chop up the DNA. Form 4 pieces Does TF X bind to DNA? Use antibody against TF X; isolate TF X using an antibody In test tube, all we have is a bunch of DNA fragments bound to TF X 2. Electrophoretic mobility shift assay (EMSA): Upstream portion of gene Y (-200 to -1) Label with 32P In test tube, take P32 probe and add in TF #1. If TR #1 is able to bind Average bp= about 1,000D x 200bp = 20,000D  piece of DNA Lets say our TF = 50,000, DNA= 20,000 and if bound together, the piece should be about 70,000 So if run out in gel, we should see all our DNA and TF if bound to it Shifting size of DNA if TF is boundPic shows us 1,2,3,4,5,6 not 7,8 bound to TF Structure of chromatin – nucleosomes2 forms:Heterochromatin- condensed formEuchromatin- open form, access to DNADNA wrapped around proteins called Histones1. Acetylation- neutralizes the charge on Lys and opens conformation. Maintains the + charge2. Methylation- keeps it closed in heterochromatin state3. Phosphorylation- open state. 2 extra – negative charges To control translation after transcription, can use ~20-26bp short RNAs:1) Those that repress translation of specific mRNAs (called microRNAs or miRNAs) 2) Those that cause mRNA degradation (termed short interfering RNAs or