mol bio exam 1

first step of initiation

tbp binds to tata box w TFIID, which changes the DNA conformation to attract RNA polymerase

after TFIID and TBP bind, what happens in initiation

TFIIE, TFIIH, and TFIIF come together with RNA polymerase. TFIIH = helicase

what TF phosphorylates Ser 5

mediator

last step in initiation seq

the ctd undergoes a conformational change when ser 5 is phosphorylated —> rna pol ii releases the gen TFs

enhancers

• where activator binds —> interacts w mediator protein in loop to start transcription

• regulatory seq that boosts transcription efficiency

what happens immediately after initiation begins

pauses after 30-50 bp and ser 2 is phosphorylated, changing rna pol configuration and initiating elongation

supercoiling

dna helicase creates positive coiling ahead of rna pol to mitigate negative coiling before —> prevents tension in DNA strand

termination

ctsf and cspf fall from rna poly to to aauaaa seq and cleave rna pol.

exo nuc degrades until it reaches rna pol, pushing it off. adds poly a tail

what do pioneer TFs do

bind even when DNA is in a nucleosome and reposition the nucleosome so that other TFs can bind

also mediate DNA bending to increase accessibility

how do transcription factors affect nucleosome positioning

recruit chromatin remodelers

types of positive TF interactions

cooperative

coocupancy

cobiding

heterodimer

types of negative TF interactions

stronger vs weaker activator

activator vs repressor

enhancer reporter

shows whether enh is sufficient to initiate promoter seq

PCR

amplify using spec primers and dNTPs

prod more product

gel electrophoresis

shows that the gene is expected vs unexpected size

illumina

what gene are we looking at

amplify dna

use spec or general primers

rna se

mrna —> fragmented cDNA —> sequence fragment ends —> map to ref genome

• shows diff gene exp + loc of upregulation vs downregulation

chip seq

cross link bound proteins to dna —> fragment dna —> precip chromatin w antibodies —> reverse cross ligate protein —> ligate adaptors and construct frag library

• shows where proteins bind to DNA and maps + and - to see overlap of tf binding site

bisulfite seq

treat dna w bisulfite to convert c to u —> leave 5-methylcytosine unaffected —> pcr/ sanger or illumina seq —> align to published genome (methylated sites = CG)

• shows where methylation has occured on genome

ATAC-seq

transposons attach to regions w euchromatin —> adaptive primers bind and fragment dna —> seq segments and align to ref genome to show open regions —> peaks indicate regions w open chr (active promoters, enhancers, and tf binding sites)

emsa

detects protein dna interactions

what does dna methyl transferase do

cytosine —> 5 methyl cytosine

• adds methyl to most CpG sites

dNMT3 vs dnmt1

dnmt3 adds to unmethylated regions in fertilization

dnmt1 adds to hemimethylated in mitosis

demethylation paths

TET oxidizes 5MC

1. prevents dnmt1 from recognizing methyl —> replication dependent dilution

2. TDG excises oxidized 5mC and repairs DNA w C

nucleosome composition

H3/H4 tetramer and H2A/2B dimers

• 147 BP DNA wound 1.65 times around core histones

• H1 linker which isn’t part of core

• H3/H4 have super modifiable histone tails

where does dna methylation take place

CpG islands of repressed gene’s promoters + repetitive regions w

what protein recruits writers

dna methyl binding protein

what stops spreading

boundary elements (ex: nuclear pore, insulators, and enzyme diffusion)

insulator

boundary protein that operates directionaly and loops to allow gene expression (ex: CTCF)

prokaryote diffs from eukaryotes

coupled transcription and translation; operons; holoenzyme instead of rna complex; [-35, -10] instead of TATA

• differences allow antibiotics to target prokaryotic transcription

chromatin remodeling

chromatin remodeling complex slides between nucs and moves them to create space

• histone chaperones remove nucleosomes and add histone variants to alter expression

dysregulation of genes

up/downregulation —> hypermethylation shouldn’t occur at gene or tumor will occur

• hypometh would be bad at repeat elements

how does dna methylation affect expression

1. methyl groups can j block tf binding

2. methyl groups recruit methyl cpg binding proteins (MeCPs)

   1. dnmt adds methyl

   2. MeCPs bind to methylated DNA and trigger heterochr

   3. recruit transcriptional repressors that block TF binding