mol bio exam 1

initiation of DNA transcription

1. TFIID binds TATA with help of TBP

2. TFIIB binds to position RNA Pol II

3. RNA Pol II, TFIIH, and TFIIE bind

   1. TFIIH unwinds DNA and phosphorylates Ser 5

4. everything disassociates and elongation begins

pausing transcription

• after 30-50 BP synth, Ser2 phosph

elongation transcription

bc Ser 2 phosph, RNA pol begins work

termination

cstf and cspf move fromm RNA pol —> RNA

• cleave and terminate seq via PAP and poly A BP

• exonuc runs bc 5’ not capped and dislodges RNA Pol II

parts of a transcription factor

1. proteins that directly bind to a specific DNA seq through DNA binding domain

2. activation domain regulates level of transcription by recruiting RNA Pol II or transcriptional cofactors

cobinding

intermediary facilitates binding of TFs

co-occupancy

rapid alternating

heterodimers

bind together

cooperative binding

binding of one facilitates binding of the other

chromatin mediated tf interactions

1. pioneer TFs remove/ displace nucleosomes

2. nucleosomes block TF binding

3. TF binding mediates DNA bending

enhanceosome model

everything matters: loc,spacing

billboard model

flexible

DNA methyltransferase

• methylates cytosine to 5-methyl cytosine

  • often at CpG sites

Dnmt 3a/3b vs Dnmt 1

denovo vs maintenance methylation

demethylation

• no direct mechanism

• TET oxidizes 5mC —> adds OH to Me to prevent maintenance methylation

  • thymine DNA glycosylase excises the incorrect base via base excision repair

methylation

highly methylated at repetitive elements, intergenic DNA

prevent TF binding

attract MeCPs —> trigger heterochromatin

recruit transcriptional repressors

chromatin spreading

1. writer makes mark

2. reader reads it and binds to nucleosome —> recruits writer

how is chr spreading prevented

reverted by eraser

boundary elements prevent spreading: nuclear pores, insulators, enzyme diffusion

chromatin remodeling

• nucleosome sliding allows access of transcription machinery to DNA

  • chromatin remodeling complex will slide histones

  • histone chaperone removes histones

  • histone replacement will replace w easier to move histones

  • histone modifying enzymes can add acetylation/ methylate differently to open up heterochr

insulators

insulator proteins bound to insulator DNA regions prevent heterochr spread and bring enh close to promoter via DNA looping

• CTCF = insulator in humans

  • can form homodimers and multidimers

  • peaks will be inside facultative chr. peaks, showing that spreading stops

  • can isolate active regions from repressed

variegation

• position of gene relative to heterochr alters expression

• when heterochr shifts closer to gene, it increases variegation

PCR experiment

• amplify DNA and visualize it

• must use a specific primer (forward and reverse)

• mutant —> no product or change in size

• doesn’t evaluate expression well

illumina

goal: amplifies DNA + seq it

• makes copies in cluster

• seq w/ specific or non-spec primers

ChIP-seq

goal: identify DNA bound by DNA binding molecule (ChIP-qPCR is used for TF binding to specific DNA seq and ChIP seq is for TF binding to all bound DNA seq)

interpretation: intersection between pos and neg site = TF binding site

procedure

1. cross link bound proteins to DNA

2. isolate chromatin and shear DNA

3. precipitate chr. w protein spec antibody

4. reverse cross link and digest protein

5. ligate p1 and p2 adaptors to construct frag library

6. illumina seq

RNA Seq

goal: method to quantify RNA

interpretation: compare infected reads to control reads

• PCA: similarities cluster and differences are far apart

• volcano plot shows up and downregulation

process:

1. make cDNAs from mRNA w reverse transcriptase

2. shatter into fragments

3. map reads

bisulfite seq

goal: evaluate methylation

interpretation: methylated C remain as C

process:

1. treatment of DNA converts unmethylated C—> U

2. PCR/sanger or illumina seq

3. uracil will dilute out to thymine over time w pcr