shift in CTD phosphorylation during elongation process

shift from phosphorylation at S5 to phosphorylation at S2

5' processing factors associated with phosph at S5

3' processing factors associated with phosph at S2

Pol II CTD phosphoylation involved in what splicing processes

exon definition (only for internal exons--needs more than one intron to identify)

exon juxtapositioning

interaction of general splicing factors

and gene specific splicing factors

which bind where

general factors (spliceosome) bind to intron

specific facors (SR) bind to exon--ESE Exonic splicing enhancers

ESE 

Exonic splicing enhancers

SRs bind

SR proteins

bind to ESE via RNA binding domains

have lots of serine & arginine amino acids

have protein-protein interaction domains

mediate the cooperative binding of U1 and U2 to specific points on intron through series of protein protein interactions that span the exon

cross exon recognition complex

interaction of SR prots and other splicing factors

allows for precise specification of exons within long pre-mRNAs

alternative splicing cascade in drosophila sex determining genes

1. sex lethal

2. transformer

3. doublesex

sex lethal

first gene in drosophila alternate splicing cascade of sex determining genes

there is a stop codon in exon three

sxl protein causes silencing of this exon

females skip this exon, males do not

as a result, only females produce functioning sex lethal protein

transformer gene

second gene in alternative splicing cascade of sex determining genes

exon two has a stop codon in it

sxl protein causes females to skip this exon

as a refult, only females produce functioning tra protein

tra protein

is an SR protein

only females make a functioning tra protein

allows for (with other proteins--Rbp1 and Tra2) exon four recognition in double sex gene

males who do not have tra protein, skip this exon for their double sex gene

double sex gene

the protein that it produced depends on the presense of absense of tra protein (an SR protein)

females express tra protein which helps recognize an exon in the double sex gene which is skipped in males

males and females produce two different double sex genes with different exons

proteins made by this gene are transcription factors--repress genes required for development of opposite sex

how does female double sex protein know to cut off before the last exon is reached?

poly(a) site

stepwise assembly model of preinitiation complex

1. TBP binds to TATA box (bends DNA)

2. TFIIB

points in direction of transcription

3. TFIIF and Pol II come together

4. TFIIE

5. TFIIH

TFIIH

ten subunits

helicase activity

DNA repair activity

phosophorylation activity (phosphorylates CTD)

xeroderma pigmentosum

form of cancer caused by disfunction of TFIIH protein (part of transcription preinitiation complex)

TFIIH is involved in recognizing polymerase stalled at damaged base pairs and in recruiting DNA repair proteins to problem sight

people with this mutation have extreme sensitvity to sunlight (common cause of DNA damage); have high incidence of cancer

telomeric silencing mechanism

RAP1s bind to telomere ends

SIR3 and SIR4 bind to RAP1s and ALSO to nearby hypoacetylated histone tails

SIR2 binds to SIR4

SIR2 is a deacetylase

silencing ok

Arginine can't be acetylated

both amino acids postive

silencing is defective

glycine has a neutral charge

what blocks binding of SIR3 and SIR4 to histone tails?

hyperacetylation

ChIP steps

1. cross link chromatin

2. isolate and shear

3. add antibody specific to certain modifications

4. immunoprecipitate

5. reverse cross link to release DNA

6. PCR

repressor directed histone deactetylation

the DNA binding domain of Ume6 binds to the URS

the repressor domain binds co-repressor Sin3

sin3 forms complex with other co-repressor Rpd3

Rpd3 is a de-acetylase

activator directed histone hyperacetylation

Gcn4 binds to URS via DNA binding domain

activator domain binds complex with subunit Gcn5, which has acetylation activity

TATA binding protein

interacts with minor groove; bends DNA

TFIIB

asymetric

N term domain points in direction of transcription

unwinds DNA using energy from ATP hydrolysis

TFIIE

docking sight for TFIIH

mediator complex

in humans, has around 30 subunits

directly helps in assembly of preinitiation complex

bridge between activators and pol II

possible that it brings pol II to start sight

telomeres of every yeast chrom behave like silence sequences

when a gene is placed within a few kilobases of any yeast telomere, it is repressed

RAP1

bind silencer sequences

and telomeres

recruits SIR3 and SIR4 and in turn SIR2

how does acetylation reduce chromatin condensation

acetylation of lysine netralizes the positive charge of lysine, eliminates their interaction with DNA phosphate groups

what histone tail state is required for silencing?

hypoacetylation

RPD3 is a

HDAC--histone deacetylase