Proteins That Regulate Transcription RR9-11 PDF

Proteins That Regulate Transcription RR9-11 Holly J | BIO200  Housekeeping Midterm : Results should come out soon Quiz 4: Opens Saturday November 2nd, closes Tuesday November 5th NO CLASS MONDAY Questions about quiz grading? Direct them t...

Proteins That Regulate Transcription RR9-11 Holly J | BIO200  Housekeeping Midterm : Results should come out soon Quiz 4: Opens Saturday November 2nd, closes Tuesday November 5th NO CLASS MONDAY Questions about quiz grading? Direct them to [email protected] SciLearn: TAs cannot send you the answers! If you can’t make the Tues/Fri slots, come prepared to discuss your solutions in post-tutorial Office Hours  Transcription From Last Week 1. Initiation Polymerase binds to the promoter sequence, locally denatures the DNA and then catalyzes the first phosphosdiester linkage 2. Elongation Polymerase moves away from the transcription start site and flips into a very processive configuration 3. Termination Polymerase recognizes stop site and releases the completed RNA, then dissociated from DNA  Transcription From Last Week Transcription Start Site (+1) Promoter: Region of DNA to which proteins bind to initiate transcription Enhancers: DNA elements that affect the ability of RNA Pols to transcribe a given gene Proximal vs Distal Upstream (in the opposite direction of transcription) vs Downstream  Transcription From Last Week Eukaryotes have 3 well conserved, multimeric Polymerases RNA Pol I: responsible for almost all rRNA in the cell RNA Pol II: synthesizes mRNA, snRNAs, siRNAs, miRNAs RNA Pol III: synthesizes tRNAs and ribosomal components  Promoters TATA Box: Sequence rich in Ts and As located 10-35 bps upstream of the TSS (+1) TBP: TATA-box binding protein - binds to the minor groove and disrupts the double helix, required for efficient Pol I/III transcription Proximal Promoter Elements & Enhancers Proximal-Promoter Elements: (6-10 bps) Transcription control elements that must be close to the promoter to influence transcription Enhancers: (50-200 bps) Transcription control element which can stimulate transcription from a promoter tens of thousands of base pairs away  Mediators Multisubunit co-activator complex (not a general TF, but involved in transcription) Bridge large sections of chromatin to enhance transcriptional initiation Interacts directly with Pol II Bind to activation domains in activator proteins Communicate regulatory signals from DNA binding TFs to RNA Pol II RNA Pol II General Transcription Factors (TFs) Required for transcription of most genes transcribed by RNA Pol II TFIID: largest general TF needed for RNA Pol II mediated transcription Made up of the TBP and TBP- associated factors (TAFs) TAFs: initiate transcription from promoters that lack TATA box  Preinitiation Complex (PIC) 1. TBP binds to TATA box promoter forming a saddle shape and interacting with the minor 1 groove of DNA 2. TFIIA associates with TBP and DNA upstream of the TBP-TATA complex, TFIIB clamps onto C-term of TBP, contacting the 2 major groove of DNA on both sides of the TATA box 3 3. Complex of Pol II and TFIIF associate with TATA-TFIIA-TFIIB complex to form the core PIC  Preinitiation Complex (PIC) 4. N-terminal of TFIIB is inserted into the RNA channel exit, stabilizing the complex and 4 holding down DNA near the TSS 5. TFIIE encloses the template DNA in a 5 protein channel over the TSS region, stabilizing the complex 6 6. TFIIH docks onto TFIIE, forming a closed PIC  Initially Transcribing Complex 7. Helicase activity in TFIIH uses energy from ATP hydrolysis to help unwind DNA at the TSS, allowing Pol II to form an open complex where 7 DNA is melted to allow for binding of template strand to Pol active site 8 8. N-term of TFIIB is released from RNA exit channel as transcription progresses 9 9. TFIIH Kinases phosphorylate the Pol II CTD  TFIIH Only TF in this process that has enzymatic activity Helicase important for initiation = XPB Kinases in TFIIH are responsible for CTD phosphorylation, which is necessary for elongation Also involved in nucleotide excision DNA repair (therefore heavily transcribed regions are repaired more effectively)  TFs are Modular! Having discrete, individual units that overall increase the function of the whole system  TFs Recognize Specific DNA Motifs! Principles of specific DNA-protein interactions were first discovered in bacteria Recognition Helix: Alpha helix from which most of the AA side chain that contacts bases in the DNA (through non covalent interactions in the major groove) extend from  Homeodomains TF Structural Motifs 60-residue (AA) DNA binding motif These proteins are often involved in development (ex Pax3, HOX family)  Zinc Fingers TF Structural Motifs Polypeptide chain folded around a Zinc ion, producing a compact domain C2H2: most common DNA binding motif in the human genome, 2 cysteines and 2 histidine side chains bind one Zinc ion C4: 4 cysteines in contact with Zinc C6: 6 cysteines bind 2 Zinc ions  Leucine Zippers (bZip) TF Structural Motifs Contain a hydrophobic residue at every 7th position in the C term sequence C terminus: coiled coil dimerization region N terminus: DNA binding domain  Basic Helix-Loop-Helix TF Structural Motifs Similar to bZip, but characterized by 2 alpha helices, connected by a short loop N terminus alpha helix: basic residues that interact with DNA Middle loop region C terminus: hydrophobic AAs spaced out like amphipathic helices (h-philic at every 2 /3 nd rd position, h-phobic at every 3rd/4th position) Combinatorial Diversity & Cooperative Binding Increase the level of regulatory diversity and stringency TFs such as bZIPs and bHLHs can exist as heterodimeric combinations of monomers Formation of dimers allows the activation domains of each monomer to be brought together in alt combos PPIs between structurally unrelated TFs in close proximity can impact protein binding affinity Linker Scanning Mutagenesis Methods Asses the importance of different parts of the regulatory regions of DNA Introduce small overlapping mutations (scrambled DNA) into region of DNA fused to a reporter gene, and asses the effect of mutation on gene expression  EMSA Methods Electrophoretic Mobility Shift Assay Detect protein-DNA complexes through use of a radiolabeled DNA probe and fractionated protein samples Cannot tell you the sequence/identiy of the bound protein(s)  In Vivo Transfections Methods Testing the ability of a protein of interest to activate or repress transcription in vivo Usually done after a TF has been identified, isolated and purified  ChIP Methods Chromatin Immunoprecipitation Antibody-based method of assessing DNA- protein binding interactions in vivo 1. Crosslink DNA with formaldehyde 2. Shear DNA 3. Immunoprecipitate 4. Sequence the bound DNA 5. Determine where DNA is bound RR11: Molecular Mechanisms of Transcriptional Initiation/Activation  What does increased transcription look like? In vivo technique to look only at actively transcribed RNAs (not degraded RNAs) Add in sequence at the 5’ region of your gene of interest, that is recognized by a protein (fused to a reporter protein) As the gene is transcribed, so will this 5’ region, which will be bound to the reporter protein  Transcription occurs in bursts! Transcriptional initiation from highly transcribed genes occurs in bursts of multiple initiation events separated by periods of no transcription Initial experiment looked at the SNAIL gene in Drosophila, which had a strong enhancer, sna, downstream of its promoter RNA Structure Reporter  Transcription occurs in bursts! Expression of reporter increased and decreased over time -> pointing towards a Burst Hypothesis over a Flux Hypothesis Frequency of burst correlates with efficiency of transcription (strength of enhancer)  P-granules Proteins involved in transcription often colocalize in puncta Think « oil in water » Transient expression  Questions? (Including SciLearn)  Know the Difference! RNA Polymerase I , II and III Northern, Southern and Western Blot ChIP vs EMSA PCR vs qRT-PCR