chromatin, epigenetics, and rna processing (22-24)

lectures 22-24

how do saccharomyces cerevisae control mating type?

• they have HMLalpha and HMRa loci that are silenced

• when time for mating, the MAT locus takes on either a or alpha configuration by non-reciprocally combing HMLalpha or HMRa

• mating type only expressed when in MAT locus

a sign that told us histones impact repression?

histone mutations resulted in expression from regions that should be silent

proteins essential for silencing the silent mating type loci

• RAP1

• SIR1, 2, 3,4

RAP1

• binds to silencer region near HMLalpha or HMRa

• also binds to repeptive sequence in telomere

• recognized by SIR proteins

SIR1

• silent info regulator

• works with RAP1

acetylation of histones tails

• reduces the interaction between histone and DNA(PO4 backbone specifically)

• neutralizes lysine(positive) residues

SIR3 and 4

bind to hypocacetylated histone tails

SIR 2 (4)

• recognize SIR3,4 and join complex

• makes histone bind tighter to DNA

• forms large complex with telomeric dna

• histone deacetylase

hypoacetylation of histone tails

• due to feedback loop of SIR proteins

• SIRs will recognize deacetlyaed regions and come join, spreads deacetlyation

Ume6

• critical dna binding protein

• has Rpd3/Sin3 corepressor complex that changes chromatin near URS

URS

dna binding transcription repressor

Rpd3

• histone deactylase

• subunit of Ume6

HATs

• histone acetyl transferases

• open up chromatin and make it more accesible

can activation domains trigger chromatin condensation?

yes

VP-16

transcription activation domain that results in dedcondensation when added to cell

pioneer transcription factors

• can only interact with exposed dna on the outside of the nucleosome

• cause dna to unwind

• recruit enzymes that change the shape of histone tails

which histones are most important/unimportant?

H1 unimportant

H3, H2, H4 very important

exampels of epigenetic traits that get passed on within the body

• inactive x chromosones

• developmental restrictions

• imprints

epigenetic readers and writers

• can sometimes be the same protein

• readers recognize marks of histone tail modifications and recruit writers after cell divison

• writers write in the same marks into new dna

CTD of rna pol II

• very extended and unstructured with weird conformations

• has YSPTSPS repeated sequence

when is ser 5 of CTD phosphorylated? by who?

after intiation but before elongation

cdk7

why does the rna pol II pause after intiation?

to give time for ser5 phosphorylation and for factors needed for later to associate

what post transcriptional processing does mRNA go through

• 5’ 7-methylguanylate cap

  • protects from exonucleases

• methylation

  • stabilizes and faciliattes nuclear transport

  • recognized dby translational factors

• polyadenylation

what happens to the CTD during the pause after intiation?

• first, ser5 phosphorylated by cdk7

• capping enzyme uses phosphorylated ser5 as a scaffold and caps 5’ end

• cdk9 phosphorylates ser2, DSIF, NELF and other enzymes

cdk9

• phosphorylates ser2 and other enzymes associated with rna pol II

• recruits splicing factors, polyadenylation factors, aexport factors

• phosphorylates NELF and DSIF

NELF (3)

• negative elongation factor

• protein that actively blocks elongation and causes stalling

• phosphoruylation by cdk9 causes it to dissociate

• once gone elongation factors can come

DSIF

• contributes to stalling

• phosphorylation by cdk9 causes it to put pressure on rna pol II’s clamp domain

when does mrna splicing occur?

co transcriptionally

splice donor and splice aceptor

donor → GU

acceptor → AG

branch point

conserved A near the 3’ end of the mRNA

slightly upstream of pyrimidine rich region

whats the spliceosome made of?

5 snRNPs

snRNP

a snRNA (U1-U6) associtated with 6-10 proteins

U1 snRNA

• binds near spice donor site

• guided by SR proteins

U2 snRNA

• partially compliment to area near branch point A

• A is unbound and bulges out

Lariat structure

• 2’-5’ linkage forms a loop of intron

• occurs from A attacking 5’ phosphate of the intron to be removed

trans-esterification reactions

• the reactions involved with forming and resolving Lariat structures

• nucleophilic attack by A on 5’PO4 of intron

• OH on exon attacks PO4 on other exon

how are lariat strcutres resolved?

debranching enzyme cuts the 2’-5’ linkage

normal riboexonucleases come in and degrade it

self splicing introns

• group 1

• group 2

  • fold into crazy structures

  • only in mt and cp

exceptions to GU AG rule

sometimes A or C replaces the G i GU

sometimes AG → AC

how is rRNA transcribed

• in tandem rerpeats

• large 48S product made

  • cut into 18S, 5.8D, 28S in humans

• spacer regions removed by special cleavage reactions using snoRNAs

snoRNAs in rRNA cleavage

• snorna bp with pre rrna to make U bulge out

• U is vulnerable to modifications

pseudoU

• stabilizes rna

• seen in rrna and trna

4 steps of tRNA processing

1. 5’ end sequence removed

2. short segment of second loop sometimes removed

3. add 5’ CCA 3’ to 3’ end

4. modify internal bases

RNA binding protein

largely mediates deciding where an intron/exon boundary is

RRM domain bindin protein

RNA recognition motif

• one of most common RNA binding proteins that help determine exon/intron boundaries

U2AF

• binds to 3’ end of intron and says THIS IS THE INTRON BOUNDARY

• has subunits that interact with small residues near AG

exon splicing enhancers

• decorate entire exon

• promote exon joining

• help U2AF recognize the boundary

• recognized by SR proteins

SR proteins (3)

• bind to exon splicing enhancers

• helps U1snRNP recognize the 5’ end of the intron

• have RRM domains

semi detailed explanation of how sex determination in drospophilia works

• females express early sxl protein during early embryogenesis

• later in development sxl specifies splicing differences in females

• males never express sxl and as a result never express tra either

• tra specifies dsx splicing, so males and females have different dsx proteins

cellular deaminases

• turn A→I and C→U

• aka rna editing

rna editing

• results in mature mRNA not matching gene sequences

• more rare in highe rorder eukaryotes

• widespread in mt and plasmids of protozoa and plants

polyadenylation of mrna

• final step of post trancriptional processing

• does not hapen to histones

how does mrna polyadenylation work?

• AAUAA and GU sequence recognized by cleavage and polyA factors

• PAP comes in, then cleavage occurs

• PAP adds ~8nt poly A tail

• PABPN1 rapidly adds ~200A residues