Tutorial RR6-8 Transcriptional Activation Mechanisms PDF

Mechanisms of Transcriptional Activation, Epigenetics & RNA Processing RR6-8 Holly J | BIOL200 | November 8th 2023  Housekeeping Midterm Viewing Session: November 9th (registration required before midnight tonight), November 13th, both...

Mechanisms of Transcriptional Activation, Epigenetics & RNA Processing RR6-8 Holly J | BIOL200 | November 8th 2023  Housekeeping Midterm Viewing Session: November 9th (registration required before midnight tonight), November 13th, both from 6:30-8:30 Student ID is required Scantrons will not be available Midterm Regrade Requests & Rubric Challenges: Fill out the survey on myCourses if you have concerns regarding grading/adding up of points OR if you feel like a question was flawed (available until November 17th) Tutorials now cover the material from the same week  RR6: Mechanisms of Transcriptional Activation and Initiation  The Mediator Enhances the interaction of key players in eukaryotic transcription Made up of multiple subunits that interact with activation domains Flexibility of domains allows the Mediator to bridge together cis- acting elements to RNA Pol II Chromatin Loops! Induce formation of PIC  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  Dynamic Kissing Model Enhancers dynamically kiss promoters along the surface of transcriptional condensates Bursts in transcriptional activation may correlate with the formation and dissolution of P-granules RR7: Chromatin, Epigenetics and the Histone Code  DNA Packaging DNA is associated with roughly the same mass of proteins -> chromatin DNA associates with histones, making the nucleosome with tails Heterochromatin - condensed, transcriptional inactive (near centromeres & telomeres) Euchromatin - Less condensed, accessible to transcriptional machinery  Silent Mating Type Loci Example of chromatin-mediated transcriptional repression Mating type in yeast is controlled by 3 loci on Chromosome III MAT: central mating type, actively transcribed HML/HMR: near left and right telomeres, transcriptionally silent copies of a or α Through non-reciprocal recombination, HMR OR L mating type is transferred to MAT locus Silencing proteins make DNA inaccessible Hypoacetylation: Induces chromatin compaction, and therefore transcriptional silencing  Epigenetic Marks Modifications on the tails of H3/H4 can be generally attributed to chromatin states Epigenetic Trait: stably heritable phenotype resulting in changes to the chromosome without altering the DNA sequence itself Epigenetic Writers: Introduce chemical medications on DNA/histones Epigenetic Readers: Specialized domain containing proteins that recognize and interpret the modifications ChiP against Histones  Histone (De)Acetylation Transcriptional activators recruit histone acetyl-transferases (HATs) neutralizes electrostatic interaction between N-term of histones and DNA backbone, allowing for the formation of complexes Transcriptional repressors recruit histone deacetylase complexes (HDACs)  Pioneer Transcription Factors DNA binding transcriptional activators that interact with exposed sequences on the outside of a histone octamer binding energy allows for unwrapping of DNA from nucleoli surface Recruit enzymes that alter the configuration of neighbouring histones Needed for activation of genes in highly condensed states RR8: RNA Processing Pt 1  mRNA Processing Pre-mRNA to a mature mRNA Modifications at the 5’ and 3’ ends of pre-mRNA are important for stability and protection 3 major co-transcriptional steps: 1. 5’ Capping 2. 3’ Cleavage and Polyadenylation 3. RNA Splicing  mRNA Capping 5’ methylguanalate cap As the nascent mRNA emerges from the RNA exit channel and reaches length of 25 nucleotides, a 5’ cap is added by a dimeric capping enzyme that interacts with the CTD domain of RNA Pol II Coupled with elongation (exchange of NELF for PAF elongation complex in association with RNA Pol II) Protects the mRNA, facilitates nuclear export and allows the mRNA to be recognized by translation factors  CTD Phosphorylation How are only Pol II transcripts subjected to capping, polyadenylation and splicing? The length of the CTD allows multiple proteins to simultaneously associate with a RNA Pol II molecule Enzymes that add the 5’ cap associate with the pCTD Splicing and polyadenylation factors associate with the pCTD  mRNA Splicing hnRNPs (heterogenous ribonucleoprotein particles): nuclear proteins that associate with mRNAs from the time they emerge from RNA Pol II, until they translocate to the cytoplasm associate with RNAs through their RNA- binding domains regulate pre-mRNA splicing, transport mRNAs out of the nucleus, etc  mRNA Splicing Removal of introns and splicing of exons Introns are not found in mature mRNAs For short transcription units, this occurs after 3’ cleavage and polyadenylation - In longer units, splicing begins before transcription ends  mRNA Splicing The Splicesesome Consists of 5 snRNAs: U1, U2, U4, U5, U6 that transiently associate with each other and the splice site to make an active splicesesome (U2/U5/U6) U1 contacts the introns splice site border U2 contacts the branch point region (motif required for splicing) 2 Transesterification Reactions: 1. OH group at the branch point attacks the 5’ phosphate at the 1st intron residue, forming a lariat (lasso shaped structure) 2. 3’ end of exon attacks 5’ end of the following exon  Splicing Cycle 6 1. U1 and U2 interact with mRNA 2. Recruitment of U4, U5, U6 5 3. U1 and U4 exit (active) 1 4. Transesterification reactions (no net energy expenditure) 2 5. Release of splicesesome 3 and mature mRNA 4 6. Intron is degraded  Questions? Including SciLearn  Practice Questions The change in factors associated with elongation blocking (NELF) for those that induce elongation (DSIF, PAF, etc) are dependent on which vent? What factors influence the formation of liquid-liquid condensates? True or False: Cleavage and polyadenylation have to occur before mRNA splicing Why are introns removed from mature mRNAs? Describe the Flux and Burst Transcription hypotheses (and why do we believe one over the other?)