Transcript Document
Announcements 1. Today – in class problem solving: including handout/worksheet on recombinant DNA technology, putting it all together AND restriction analysis problems 2. Based on your input, I’ve compiled slides from lectures that are good overviews of different topics covered. As time allows, I will go over “big picture” and main points of topics requested – in order of “popularity”. 3. Wednesday is review day – when I take your specific questions from problem sets, end of chapter problems, etc… 4. Exam 3 is Thursday, Friday, and Sunday at testing center. You will need a bluebook, pencil, calculator, ID – as usual. 5. GRE workshop Nov. 13 - Bovee UC 7pm- detail by main office Restriction Mapping problems *Note: fragments are linearized to start with. * Figure 18.23 Learning Check Linear DNA fragment Marker EcoRI BamHI EcoRI/BamHI 15 10 9 8 6 7 5 8 6 5 4 2 What is the restriction map of this cloned DNA fragment, showing the locations of the restriction sites and relative distance between sites? Transcription Making Sense of the Strands • DNA coding strand = Sense Strand • DNA template strand = Antisense Strand • mRNA formed = Sense Strand Coding strand 5’ mRNA 5’ 3’ 3’ 3’ Template strand 5’ Prokaryotic Promoter Lies Just Upstream (5’) of Transcribed Region; RNA Polymerase Binds Two Places -35 Region -10 TATA Box Anatomy of a Eukaryotic Gene (Protein Encoding) Pol II, Basal TFs bind CAAT Box TATA Box Cis-regulatory Elements may be located thousands of bases away; Regulatory TFs bind. Transcription factors TBP-TATA binding protein TAFs- TATA assoc. factors RNA Processing in Eukaryotes Pre-mRNA (primary transcript) 5’ cap and 3’ cleavage Poly A tail added to 3 ‘end Splicing: introns removed Mature mRNA Eukaryotic vs. Prokaryotic Transcription • • In eukaryotes, transcription and translation occur in separate compartments. In bacteria, mRNA is polycistronic; in eukaryotes, mRNA is usually monocistronic. – Polycistronic: one mRNA codes for more than one polypeptide – moncistronic: one mRNA codes for only one polypeptide • 3 RNA polymerases in euk., 1 in prok. Binding of Basal Transcription Factors required for euk. RNA Pol II binding. • Processing of mRNA in eukaryotes: – 5’ 7-methylguanosine (7mG) cap added – 3’ Poly-A tail added – Splicing out of introns Problems of Multicellularity • All of our genes are present in every cell, but only certain proteins are needed. Pancreatic cell + insulin - neurotransmitter Neuron - insulin +neurotransmitter • Expression of a gene at the wrong time, in the wrong type of cell, or in abnormal amounts can lead to deleterious phenotypes or death - even when the gene itself is normal. Promoters: Eukaryotic vs. Prokaryotic RNA pol II RNA pol Promoters: sequences adjacent to genes, where RNA pol binds to initiate transcription Euk. - Chromatin and TFs Prok. - Naked DNA and no TFs needed Eukaryotic Enhancers and Promoters Promoters- needed for basal level transcription Enhancers- needed for full level transcription; location and orientation variable Two types of transcription factors bind enhancers and affect levels of txn: true activators and anti-repressors Combinatorial Model of Gene Expression Liver Brain Regulatory TFs increase transcription activity No reg.TFs in this cell for albumin enhancer Binding of True Activator TFs to Enhancers Greatly Stimulates Transcription Looping of DNA allows Activator TF bound to Enhancer to interact with Promoter, facilitating binding of Basal TF complex. Chromatin remodeling TFs can recruit HATs or HDs Bacterial Strategy • If glucose is present, – then use glucose as a carbon source. • If glucose is not present, and if lactose is present, – then use lactose (indirectly) as carbon source. Levels of enzymes needed to use lactose as carbon source increase dramatically when lactose is present; enzymes are inducible and lactose is the inducer. The Operon Model - components pol R L Repressor protein has 2 key binding sites pol R R pol pol R R L L pol pol R pol R pol Learning Check pol R L Will transcription and translation of Z, Y,and A enzymes occur? What would happen if a wild-type copy of I was added? Catabolite Repression of lac Operon -/+ Glucose Goal: efficiency, don’t waste energy converting lactose, when glucose available Tryptophan Operon - Repressor Binds when tryptophan is present Other Theoretical Possibilities Separation of Nucleic Acids by CeCl Gradient Centrifugation DNA Labeling with 15N Meselson-Stahl Experiment Subsequent Generations Labeled with 14N Cesium Chloride Gradient Banding Expected Results From Conservative or Dispersive Reproduction If Conservative: Two bands, heavy and light, in 1st and 2nd generations If Dispersive, one smeary band in 1st and 2nd generations Expected Results if Semiconservative These results were obtained. A related experiment was performed in plants (Fig. 12.5) Steps of DNA Synthesis • Denaturation and Unwinding • Priming and Initiation • Continuous and Discontinuous Synthesis – Including Proofreading and Error Correction • Removal of Primer • Ligation of nicks in backbone Continuous and Discontinuous Synthesis • Continuous on Leading Strand. • Discontinuous on Lagging Strand creates Okazaki fragments. • DNA ligase joins nicks in backbone. IV. The Eukaryotic Problem of Telomere Replication RNA primer near end of the chromosome on lagging strand can’t be replaced with DNA since DNA polymerase must add to a primer sequence. Do chromosomes get shorter with each replication??? Solution to Problem: Telomerase • Telomerase enzyme adds TTGGGG repeats to end of lagging strand template. • Forms hairpin turn primer with free 3’-OH end on lagging strand that polymerase can extend from; it is later removed. • Age-dependent decline in telomere length in somatic cells, not in stem cells (germ cells), cancer cells. Aminoacyl tRNA synthetase Special Anticodon-Codon Base-Pairing Rules Overview of Prokaryotic Translation 1 2 EPA 3