Chapter 9: Transcriptional Regulation and Epigenetics

What is responsible for negative transcription control in E. coli

lac operon

Who discovered lactose metabolism

Jacob & Monod (1950)

What controls transcription in E. coli

Operator (o) controls transcription

What happens in E. coli in lactose (-) situations

repressor (i) binds (o); inhibits transcription initiation

What happens in E. coli in lactose (+) situations

allolactose allosterically inhibits repressor, permits operon expression

What 3 things are expressed in E. coli’s operon

1. β-galactosidase (cleaves (1,4) glycosidic bond in lactose (glucose + galactose)

2. Permease

3. Transacetylase

What is responsible for positive transcription control in E. coli

Glucose

What is E. coli’s preferred Carbon source and what does it do

Glucose; represses lac operon

What happens in E. coli in Glucose (-) situations

adenylyl cyclase converts ATP to cAMP; cAMP allosteric CAP activation (catabolite activator protein)

What does cAMP / CAP interact with

RNAP α subunit; binds promoter

What happens in E. coli in Glucose (+) situations

α-ketoglutarate (3-c Krebs intermediate) inhibits adenylyl cyclase & lac operon

What is cis-Acting Regulatory Sequences HSV Thymidine Kinase Promoter

Herpes Simplex Virus

Core promoter elements & 3 additional upstream elements required for transcription

• CCAAT box (-75)

• 2 GC boxes (GGGCGG) (-50, -100)

What is cis-Acting Regulatory Sequences SV40 Promoter and Enhancer

simian vacuolating virus 40

Promoter: TATA box, 6 GC boxes

Upstream Enhancer: 2, 72 bp repeats

What are Enhancers

regulatory sequences often long distances from +1 site (e.g. 10 kb)

What do Enhancer sequences do

bind different regulatory proteins

What are some functions of Enhancers

Tissue specific gene expression: development, differentiation, response to hormones & growth factors

What is the Action of Enhancers if

1. No Enhancer

2. + Enhancer

3. Different Enhancer location / orientation

1. basal levels of transcription

2. stimulates expression (stimulated transcription)

3. no effect on activity, still have stimulated transcription

Enhancers act via…

trans-acting factors

What does DNA looping allow for

allows interactions between GTFs, Enhancer binding proteins (specific transcription factors), & mediator

What protein stabilizes looped DNA

Cohesin

How are Chromosomal Domains formed

loops (100-1000 kb) organize chromatin into functional domains

What is the function of Chromosomal Domains

prevents promoter interaction with inappropriate enhancers

How are Chromosomal Domains maintained

maintained by interaction between CTCF (CCCTC-binding factor) & Cohesin

Specificity Protein 1 (Sp1) is an example of a ____ and it binds ____

Transcriptional Activator, GGGCGG (GC boxes)

What are the domains in Transcriptional Activators and what do they do

• DNA binding domain binds enhancer elements

• Activation domain stimulates transcription (Mediator / GTF interaction) and interact with coactivators to modify chromatin

Activator and coactivators ____ chromatin while Repressors and corepressors ____ chromatin

relax, tighten

What is the main function of Eukaryotic Repressors

Bind DNA and inhibits transcription

What are the 3 mechanisms of Repressors ability to inhibit transcription

1. block activator binding or prevent RNAPII interaction

2. inhibit transcription by interaction with GTFs and Mediator

3. act through co-repressors to alter chromatin structure

Structure and Function of Transcriptional Activators

• stimulate transcription through regulatory activity

• DNA binding domain; transcriptional activation domain, interacts with other proteins / molecules (e.g. estrogen receptor)

How is transcription elongation initiated

phosphorylation of CTD Ser-5

What happens to the RNAPII about 50 nts from the TSS

it is paused mediated by NELF and DSIF

(Negative elongation factor and DRB sensitivity inducing factor)

When RNAPII gets paused ~50 nts from the TSS, how does it get unpaused

pTEFb phosphorylates CTD Ser-2, NELF, & DSIF; NELF dissociates

(positive Transcription Elongation Factor b)

Productive elongation is characterized with

association of CTD associated processing factors

What size is transcriptionally active chromatin

relatively decondensed, 30 nm fiber

Transcriptionally active chromatin undergoes ____

modification

What types of modification does transcriptionally active chromatin undergo

histone and nucleosome modification

Transcriptionally active chromatin is associated with…

non-histone proteins (GTFs, Replication, Repair Proteins)

How is chromatin loosened to become transcriptionally active

H3 Lys acetylation neutralizes (-) charge, relaxes chromatin structure

Chromatin condensation / repression is mediated by

(tightens chromatin so that it is not transcriptionally active)

H3 Lys methylation via repressor proteins

What is the function of the H2A, H2B, H3, & H4 domain of histone

histone interaction, DNA binding

What N-terminal tail modifications can histone undergo

1. acetylation

2. methylation

3. phosphorylation

4. ubiquitination

What are transcriptional activators and repressors associated with histone proteins

activator: HAT (histone acetyltransferase)

repressor: HDAC (histone deacetylase)

Chromatin is ____ free to allow transcription factor binding

nucleosome

Chromatin has ___ sensitive sites

DNase

What is the characteristic of nucleosomes flanking promoters

H3K4me3 (trimethylated H3 Lys-4); enhancers

What do chromatin remodeling factors do and how

what are they associated with

alter transcription factor access (ATP-dependent)

alters nucleosome position, chromatin conformation, and ejects nucleosome from DNA

associated with CTD elongation factors to alter DNA access by RNAPII