lec27 Flashcards
How and why does TBP bind the TATA box?
TBP binds the minor groove of the TATA box using hydrophobic phenylalanine residues that wedge into the DNA. This bends and distorts the DNA, making it easier for RNA polymerase II and other transcription factors to assemble at the promoter.
What are the roles of mediators, transcription factors (TFs), and activators in RNA polymerase II (RNAP II) transcription initiation?
Mediators help in recruiting RNA polymerase II (RNAP II) to the promoter and facilitate communication between activators and RNAP II.
Transcription Factors (TFs) bind to specific DNA sequences in the promoter to help assemble the transcription machinery and initiate transcription.
Activators are proteins that bind to enhancers or other regions to increase the efficiency of transcription initiation by helping recruit RNA polymerase II and TFs.
Chromatin Remodeling happens first to make DNA accessible before these factors can bind to initiate transcription.
enhancers
Enhancers are regulatory DNA sequences that activator proteins bind to. They help increase transcription by assisting the assembly of RNA polymerase II and general transcription factors at the promoter—often through mediator proteins. The more activators bound to enhancers, the stronger the transcriptional activation (i.e., higher gene expression).
What is the function of chromatin remodeling complexes (CRCs), and how do key transcription elements interact with DNA?
CRCs move or loosen nucleosomes to expose DNA for transcription.
TAP (Transcription Activator Protein) binds to the enhancer sequence.
TBP (TATA-binding protein) binds to the TATA box.
This coordinated interaction helps recruit RNAP II to the promoter.
hat is the purpose of phosphorylation on the CTD of RNA Polymerase II?
Phosphorylation of the CTD (C-terminal domain) allows RNA Pol II to initiate transcription and recruit mRNA processing enzymes for 5’ capping, splicing, and polyadenylation.
mrna capping
5’ cap is added early on, before synthesis of the primary
mRNA transcript is complete
* only added to RNA polymerase II (mRNA) transcripts
* 5’ cap participates in binding of mRNA to ribosome to
initiate protein synthesis
* may also stabilize mRNA by protecting it from nucleases
How does mRNA processing happen during transcription?
Phosphorylation of the CTD of RNA Pol II recruits processing enzymes—capping, splicing, and polyadenylation factors. These enzymes act on the mRNA as it’s being synthesized, allowing co-transcriptional processing.
What happens after RNAP II transcribes the AAUAAA signal?
An endonuclease cuts the RNA 10–30 nt downstream of AAUAAA, and polyadenylate polymerase adds ~80–250 A’s to form the polyA tail, helping with mRNA stability, export, and translation.
How was splicing first discovered using R-looping? and what is cdna and its signifcance
Scientists hybridized mature mRNA to ssDNA from its gene. Introns in DNA didn’t match mRNA, forming visible loops under an electron microscope — proving introns get spliced out cDNA is made from mature mRNA. Comparing gene DNA to cDNA shows introns are missing in mRNA, confirming splicing.
Q: What is the spliceosome and how does it work?
The spliceosome is a complex of snRNPs (small nuclear ribonucleoproteins) that removes introns from pre-mRNA. It recognizes splice sites and the branch point A, forms a lariat structure, and joins exons together. The 2′ OH of branch point A attacks the 5′ splice site, forming the lariat, and then the free 3′ OH of exon 1 attacks the 3′ splice site to join the exons.
two jobs of phosphorylation on poymerase ii?
to start transcription and as a landing base (CTD) for capping enzyme
What are snRNPs and what do they do in splicing?
snRNPs (small nuclear ribonucleoproteins) are made of snRNA + proteins. They base pair with pre-mRNA at splice sites and help form the spliceosome, which catalyzes the two transesterification reactions to remove introns. U2 snRNP pairs near the branch point but keeps branch point A unpaired so its 2’ OH can attack.
Compare Group I and Group II self-splicing introns.
Group I:
Needs free guanosine (GMP, GDP, GTP, guanosine) as a cofactor (not guanine!)
G’s 3’OH attacks 5’ splice site
Intron released linear
Requires Mg²⁺
Group II:
Uses internal 2’OH of branch point A
Attacks 5’ splice site → forms lariat
Same chemistry as spliceosome, but no proteins needed
Also needs Mg²⁺
✅ Both are ribozymes (RNA acts as the enzyme)
✅ Both splice pre-mRNA without proteins!
What is alternative splicing and why is it important?
Alternative splicing is a mechanism that allows a single gene to produce multiple protein products by splicing pre-mRNA at different splice sites.
It occurs in ~30% of human genes, contributing to protein diversity.
By including or excluding different exons, alternative splicing generates multiple protein isoforms from one gene.
It’s regulated and can change during development or in different cell types.
Isoforms are different protein products made from the same gene through alternative splicing.
Tropomyosin and Alternative Splicing:
The tropomyosin gene produces a variety of isoforms through alternative splicing.
Different cell types express different tropomyosin isoforms, which interact with specific proteins to regulate actin filament function.
Cell-specific proteins bind to the spliceosome and determine which splice sites are used.
The diversity of tropomyosin isoforms allows for specialized actin filament populations and plays a role in both space (different cells) and time (developmental stages).
What is the very first step in eukaryotic transcription initiation?
Chromatin remodeling proteins (e.g., SWI/SNF) and histone acetylases open chromatin by sliding/ejecting nucleosomes and acetylating histones to expose the promoter.
Key: Without this, the DNA is inaccessible!
