Biology Quiz: RNA Transcription Concepts

Housekeeping

• Midterm results will be available soon
• Quiz 4 opens Saturday, November 2nd, and closes Tuesday, November 5th
• No class on Monday
• Email [email protected] for quiz grading questions
• SciLearn TAs cannot provide answers
• Students should attend post-tutorial office hours if they can't attend the scheduled review sessions

Transcription

• Initiation: RNA polymerase binds to the promoter, denatures DNA locally, and catalyzes the first phosphodiester linkage
• Elongation: RNA polymerase moves away from the start site, changing to a processive configuration
• Termination: RNA polymerase recognizes a stop site, releases the completed RNA, and dissociates from DNA

Transcription (From Last Week)

• Transcription Start Site (+1): The starting point of transcription
• Promoter: Region of DNA where transcription factors bind to initiate transcription
• Enhancers: DNA elements influencing the ability of RNA polymerase to transcribe a gene.
• Upstream/Downstream: Upstream is in the opposite direction of transcription from the start point, downstream is in the same direction.
• Proximal/Distal: Proximal elements are close to the promoter, distal elements are further away.

Eukaryotic RNA Polymerases

• Eukaryotes have 3 RNA polymerases (I, II, and III)
• RNA polymerase I is responsible for most ribosomal RNA
• RNA polymerase II synthesizes mRNA, snRNAs, siRNAs, and miRNAs
• RNA polymerase III synthesizes tRNAs and ribosomal components.

Promoters

• TATA box: Region rich in Ts and As, located 10-35 bases upstream of the transcription start site.
• TBP (TATA-box binding protein): Binds to the minor groove, disrupting the DNA double helix, crucial for efficient pol I/III transcription.

Proximal Promoter Elements & Enhancers

• Proximal promoter elements: Short (6-10bp) elements near the promoter that control transcription
• Enhancers: Elements (50-200bp) that can stimulate transcription from regions of DNA thousands of bases away from the promoter.

Mediators

• Multisubunit co-activator complex
• Bridges chromatin sections to enhance transcription initiation
• Interacts directly with RNA polymerase II
• Binds to activation domains
• Communicates regulatory signals from DNA binding TFs to RNA polymerase II

RNA Pol II General Transcription Factors (TFs)

• TFIID is the largest general transcription factor essential for RNA polymerase II-mediated transcription.
• Made up of TBP and TBP-associated factors
• TAFs initiate transcription from promoters lacking TATA box

Preinitiation Complex (PIC)

• TBP binds to the TATA box forming a saddle shape and interacting with the minor DNA groove
• TFIIA associates with TBP and DNA upstream.
• TFIIB clamps onto TBP, contacting the major groove on both sides of the TATA box.
• Complex of RNA polymerase II and TFIIF associate with TATA-TFIIA-TFIIB complex to form the core PIC
• N-terminal of TFIIB is inserted into the RNA channel
• TFIIE encloses the template DNA.
• TFIIH docks onto TFIIE, forming the closed PIC

Initially Transcribing Complex

• Helicase activity in TFIIH unwinds DNA at the TSS, enabling the formation of an open complex
• N-term of TFIIB is released as transcription progresses
• TFIIH Kinases phosphorylate RNA polymerase II's CTD

TFIIH

• Only transcription factor with enzymatic activity.
• Helicase is essential for initiation.
• XPB proteins are kinases.
• Kinases in TFIIH phosphorylate the CTD.
• Necessary for elongation.
• Involved in nucleotide excision DNA repair.

TFs are Modular!

• Transcription factors have discrete domains for DNA binding, activation/repression, chromatin remodeling, nuclear import, and protein interaction.

TFs Recognize Specific DNA Motifs!

• Recognition Helix: An alpha helix from which most of the amino acid side chains that contact DNA bases extend, primarily interacting through non-covalent interactions in the major groove

Homeodomains

• 60-amino acid DNA binding motif important in development (ex: Pax3, HOX family).

Zinc Fingers

• Polypeptide chain folds around a zinc ion.
• Most common DNA binding motif.
• C2H2: 2 Cysteines and 2 HIstidine side chains bind one Zinc ion.
• C4: 4 Cysteines bind to zinc
• C6: 6 Cysteines bind to two separate zinc ions

Leucine Zippers (bZip)

• Hydrophobic residue every 7th position.
• Coiled-coil dimerization in C terminus.
• DNA binding in N terminus.

Basic Helix-Loop-Helix

• Similar to bZip.
• Two alpha helices connected by a short loop.
• N terminus alpha helix: Basic residues interacting with DNA
• Middle loop region
• C terminus: hydrophobic AAs spaced out like amphipathic helices (h-philic every 2nd/3rd, h-phobic every 3rd/4th position)

Combinatorial Diversity & Cooperative Binding

• TFs (bZIPs and bHLHs) can exist as heterodimers (combinations of monomers)
• Dimer formation brings together activation domains.
• Protein-protein interactions between unrelated TFs can affect protein-binding affinity.

Linker Scanning Mutagenesis

• Method to determine the importance of different parts of regulatory DNA sequences.
• Introducing overlapping mutations into a DNA region.
• Assessing the effect of mutation on gene expression.

EMSA (Electrophoretic Mobility Shift Assay)

• Detects protein-DNA complexes using radiolabeled DNA probes and fractionated protein samples.
• Cannot determine the bound protein sequence.

In Vivo Transfections

• Testing the ability of a protein to activate/repress transcription in living cells.
• Typically conducted after identifying, isolating, and purifying a transcription factor.

ChIP (Chromatin Immunoprecipitation)

• Antibody-based method for assessing DNA-protein binding interactions in vivo.
• Crosslinking DNA with formaldehyde, shearing the DNA, immunoprecipitating bound DNA, sequencing the DNA, and determining the binding sites.

What does increased transcription look like?

• In-vivo technique looks at actively transcribed RNA, not degraded RNA
• Sequence added to the 5' region of interest, recognized by a reporter protein
• Transcribed gene region will also be bound by the reporter protein

Transcription occurs in bursts!

• Transcriptional initiation from highly transcribed genes occurs in bursts
• Experiment looked at the SNAIL gene in Drosophila, which had a strong enhancer (sna) downstream of its promoter.

P-granules

• Proteins involved in transcription often colocalize in puncta (dots)
• Liquid-liquid condensate formation.
• Examples of factors involved: concentration of macromolecules, valency, electrostatic interactions, post-translational modifications, intrinsically disordered proteins, transient expression

Know the Difference!

• Distinguish between RNA polymerases I, II, and III
• Differentiate between Northern, Southern, and Western Blots
• Compare ChIP and EMSA
• Distinguish between PCR and qRT-PCR