BIO 344 1st Edition Lecture 8Outline of Last Lecture I. Integrating glucose and lactose signalsII. Glucose repression is combinatoriala. cAMPb. CAPIII. Generalizing RegulationIV. Generalizing Transcriptiona. RNAPV. Stages of Transcript Initiation by RNAPVI.Outline of Current Lecture I. Stem Cell Researcha. Pluripotentb. Direct reprogrammingc. Metastable statesII. Eukaryotic Transcription—RNAP IIa. Promotersb. Enhancersc. Transcription factors III. Tethering a protein to DNAThese 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.a. Persistence lengthIV. CombinatorialCurrent LectureEukaryotic Transcription—Stem Cell research- Embryonic stem cells have differential potential—can differentiate into cells of all tissue typeso Brain, liver, fat, pancreatic beta cell, muscle- Stem cells can give rise to other cell typeso Pluripotent- Can it be reversed? Can we use one tissue type, reverse it, and make a different tissue type?o Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors No stem cell needed—skin cell straight to heart cell Use transcription factors to manipulate cell differentiation behavior- Metastable states in gene expression landscapeo Development—different areas of landscape for different cell typeso Pluripotenet reprogrammingo Lineage reprogramming—pushing a cell type to express a different subset of theirgenotypeEukaryotic Transcription—RNA Polymerase (focus on RNAP II)- Three types of RNAPo I: rRNAo II: mRNAo III: tRNA, 5s rRNA, telomerase RNA- RNAP IIo Shaped similarly to bacterial RNAP but is controlled differentlyo Composed of multiple polypeptides that come together to function Requires multiple genes to assemble- Promoters, enhancers, and transcription factorso Core promoter is immediately upstream of the geneo Regulatory factors are distant from the gene for other RNAPs to bind- Core promoter—start point of transcriptiono includes a combo of Inr and TATA or DPE Initiator element (Inr) TATA box—conserved TATA sequence- Upstream of many genes- Deletion mutants helped define TATA boxo Primer extension Reverse transcription—reading back to determine start site Make deletions of different lengths from the promoter If deletion is long enough to delete TATA box, transcription still occurs but starts at the incorrect location—RNAP positioned incorrectly DPEo Need a combination because we need 16 nucleotides to uniquely recognize a sequence one or two promoter elements is not enough to specify a gene Also need even more than the core promoter—need addition factors (transcription factors)- Transcription factorso TFIIX X is a D, H, F, E, etc,o TFIID TBP component recognizes TATA box- Symmetrical, behaves like a dimer- Binds to TATA and distorts DNAo TATA has weaker hydrogen bonding and is more susceptible to bending- Enhancerso Required for high level transcription o Act independently of orientation and positiono Upstream activatorso Ex: gal4 bound to UAS UAS is like the enhancero Gel Shift experiment Take a radioactively labeled DNA and a culture tube with yeast or human cells, grind cells, and centrifuge cells Gives an extract-- contains anything free floating, like macromolecules  Mix extract with DNA and separate DNA Bands separate, moving through gel at different rates, due to different proteins attached Looking at bands—labeled vs unlabaled- When equal distribution of labeled and unlabeled wild-types of identical sequence, gal4 binding is equally disctributed- When mutation in recognition sequence, and mutant is 10 times more available than the wild-type, still see a large distribution of gal4 bound to the wild-typeo Single nucleotide change affects gal4 binding to DNA- When mutation was between recognition sequences but not within the sequence, actually saw that gal4 had a greater affinity for the mutant than the wild typeTethering a protein to DNA- Gal4DB-UAS is a dimero Does UAS have to be close to the promoter? See table on slides—from table 2 of the paper- Tethering increases its local effective concentration- Enhancers can work at great distanceso Contrary to bacteria—CAP and O1 need to be in close proximity to O3- Eukaryotic DNA has rigidityo Persistence length= expression of rigidity per unit length Natural flexibility at a certain lengtho If the DNA strand is short, 50 base pairs for example, the two ends can’t contact each othero But if 500 base pairs long, the flexibility of the strand allows the ends to contact each other for the proteins to bindo However, if too long, the extent of flexibility is so great that the ends are less likely to come into contact with each othero If the DNA is untethered (in two pieces) the two pieces are unlikely to contact each other floating around in the cytosolCombinatorial Control- Do all transcription factors recruit RNAP II?o Combinatorial recruitment enhances likeliness of bindingo What does gal4 recruit? Gal11—a component of the mediator complex and interacts with gal4- Mediator complex= mediator between transcription factors and RNAPo Bridges interactionso Combinatorial control= enhancers regulate cell type specific transcription Certain combination of transcription factors bound to different types of enhancers to regulate cell