mol bio exam 2

TFIID

composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs), binds to the core promoter region of genes and serves as a scaffold for PIC

UAS

upstream of gene’s promoter and acts as a binding site for transcriptional activators (analogous to enhancer regions)

CstF

promotes cleavage of mRNA

CPSF

recruits PAP to end of mRNA for poly-adenylaiton

Rat1

5’ - 3’ endonuclease for Torpedo model of transcription termination in YEAST

hXrn2

5’ - 3’ endonuclease for Torpedo model of transcription termination in EUKS

S7P, S5P

promote mRNA capping (peak near the 5’ end of gene in metagene profile)

S2P

promotes txn elongation and cleavage of mRNA (peaks in the middle/end of gene) but drops immediately after PAS is transcribed

TFIIB, TFIIH (Kin 28), TFIIF (Tfg1)

GTFs found at 5’ end of genes

Cet1

capping enzyme

Pcf11

3’ end formation

Spts

elongation factors, peak in the middle of gene

TFIIH/Cdk7

promotes PIC —> OC transition, phosphorylates Ser-5 which disassembles mediator so it can bind to CTD and escape promoter

triptolide

inhibits TFIIH and prevents PIC —> OC

Cdk9/CycT

parts of P-TEFb that phosphorylates Ser-2, DSIF, and NELF so PEC —> EC

flavopiridiol/DRB

inhibits Cdk9/CycT

integrator complex

prevents PEC —> EC if right elongation factors aren’t present

TFIIS

cleaves out backtracked RNA during Pol II stall

Spt6

histone chaperone protein that associates with Pol II to reconstruct nucleosomes following transcription (prevents chromatin disruption)

promoters/enhancers = INHERENTLY bidirectional

not always seen due to regulation that degrades anything antisense

chromatin-associated RNA enrichment

purifies chromatin b/c nascent RNA is usually associated with them and b/c chromatin is so insoluble we wash it a shit ton

can map 5’ ends (determine where txn starts) or the entire gene but expect to see introns as well

Pol II-associated RNA enrichment (NET-seq)

start with chromatin, digest it, then purify Pol II

can still lead to contamination b/c random RNA can still be caught in assay (why we see a lot of tRNA and rRNA in this despite not being transcribed by Pol II)

Run-on RNA enrichment (PRO-cap/PRO-seq)

permeabilize cells to wash out NTPs and replace with biotinylated NTPs, allow for 1-2 bp elongation and pull down biotinylated NTPs —> gives you bp resolution of where the active site of RNA Pol II is

Metabolic RNA labelling (pulse/chase) aka TT-seq

feed cells 4sU and biotinylate U —> pull down and purify to find where they are added OR chemically convert 4sU and sequence because the chemical conversion will cause them to be read as diff bases (look for signature mutation to figure out nascent RNA)

• drawback for both of these is that there’s no base pair-level resolution but 4sU with chemical conversion doesn’t include any purification/pull down —> straight to sequencing

1st bond broken and made in RNA splicing

-OH in BP attacks 5’ss to make a new bond, but it breaks the bond between 5’ss and intron

2nd bond broken and made in RNA splicing

bond between intron and 3’ss is broken to make a new bond b/w 5’ss exon 1 and exon 2

U1 function

recognizes and binds to 5’ss —> popped out and replaced by other factors that bring BP closer to 5’ss to make that first bond

ATP-dependent and use proteins to help (such as helicases that peel proteins off RNA to get rid of secondary structures)

why spliceosomes are more efficient than self-splicing introns

exon definition (long introns)

5’ss recognizes upstream of 3’ss and that pair recognition allows for correct splicing (what they found in the minigene high throughput analysis)

intron definition (short introns)

5’ss and downstream 3’ss are close enough to interact and splice out intron

SMN1 gene

codes for survival motor neurons, essential for normal function and maintenance of motor neurons

IKBKAP gene

elongation factor for RNA transcription

CLIP

crosslinking and immunoprecipitation

steps of CLIP

crosslink RNA-proteins by UV, fragment RNA, add adapters to fragment and run on SDS-PAGE in both high RNase (free protein) and low RNAse (protein bound to RNA) conditions, cut out purified protein-RNA band and digest proteins, add RT and synthesize cDNA, sequence

cDNA can either terminate at location of cross-link or read-through with signature mutations

CLIP or 4sU treatment

2 ways to make library for CLIP

liquid-liquid phase separation

molecular demixing event where certain biomolecules separate from surrounding environment to form condensates

nuclear speckles and nucleoli

examples of condensates

condensate

membrane-less organelles with enrichment of particular environment

form through cooperative behavior and can be altered in stressful conditions

nuclear speckles

enriched for splicing factors and RNA processing

nucleoli

rDNA genes, ribosomal manufacturing

SPRITE

another pool-split experiment but without beads —> can use to determine RNA-DNA or RNA-RNA intxns (shows you where in the nucleus specific molecules are localized)