LS 3 - Transcription in Eukaryotes

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Research leading up to transcription in eukaryotes

1 - 1980s: focus on DNA-protein interactions in vitro (affinity chromatography, reporter gene assays, in vitro transcription assays, gel shift assays, DNA footprinting); 2 - these studies identified many sequence-specific DNA binding proteins; 3 - 1990s: it became clear that in addition to DNA-protein interactions, transcription also involved: protein-protein interactions, chromatin structure, and cellular compartmentalization

Overview of eukaryotic transcription

1 - much more complex than prokaryotic transcription; 2 - primary transcript is processed extensively to yield mRNA; 3 - mRNA is transported out of nucleus to cytoplasm for translation; 4 - each of these steps can be regulated

Chromatin and transcription

chromatin inhibits transcription in eukaryotes; 1 - in vitro, RNA polymerase and the general transcription factors are sufficient to initiate transcription (with DNA w/o nucleosomes); 2 - in vivo, additional proteins are necessary to deal with the inhibitory nature of nucleosomes (mediator complex, DNA-binding regulatory proteins, chromatin remodeling and modifying proteins)

RNA polymerases in eukaryotes

1 - RNA pol I (involved in transcribing ribosomal RNA, these are not translated); 2 - RNA pol II (involved in trascribing the mRNA, these are translated); 3 - RNA pol III (involved in transcribing tRNA and other small RNAs, these are not translated)

Assembly of active transcription complexes in prokaryotes vs eukaryotes

1 - prokaryotes: RNA polymerase holoenzyme binds directly to the promoter; 2 - eukaryotes: general transcription factors (TFFIID, TFIIA, TFIIB) must bind to the promoter before Pol II

Promoter elements in eukaryotes

1 - promoters in eukaryotes contain multiple DNA binding sequences; 2 - TATA box, upstream activating sequence, proximal promoter elements, enhancers, long range regulatory elements, insulator

TATA box

1 - sequence found at around -30 of eukaryotic promoters; 2 - contains TATA sequence; 3 - this forms the heart of the core promoter

Core promoter

1 - the "core promoter" consists of about 60bp around the transcription start site; 2 - transcription factor TFIID interacts in multiple sites along the plus region of the gene; 3 - TFIID also has a region (TBP) that reacts with the TATA box; 4 - TFIIB has a region that interacts upstream of the TATA box; 5 - promoters may contain some, all or none of these elements

TFIID

compoposed of TBP and TAF; 1 - TBP: TATA binding protein (recognizes TATA and binds there); 2 - 10 TAFs: TBP associated factors (has multiple binding sites across the promoter region)

TFIIB

1 - single polypeptide that binds to the TBP-DNA complex and stabilizes it; 2 - recruits and positions Pol II-TFIIF complex; 3 - inserts into RNA exit channel of Pol II, similar to sigma factor; 4 - helps setup transcription start site

TFIIE

involved in TFIIH recruitment

TFIIH

1 - multiple polypeptide important for melting the promoter (opening the DNA); 2 - has DNA helicase activity (ability to unwind DNA strands, needs ATP); 3 - has CTD (carboxy terminal domain) kinase activty (add phosphates to a region at the carboxyl terminus of the RNA polymerase)

Mediator

1 - transduces regulatory information from activators and repressors to Pol II; 2 - can also affect the chromatin structure

In vitro transcription assays

1 - many sequences and proteins were assayed using run-off assays: in vitro transcription; 2 - take a plasmid in which youve cloned a eukaryotic promoter; 3 - digest the plasmid at a unique restriction site (linearize it, loses supercoiling); 4 - add proteins, the polymerase and general transcription factors to the DNA and see if specific transcription would initiate from the promoter; 5 - they noticed that in addition, nonspecific transcription would occur when teh polymerase binds to the end of the DNA and transcribes the entire plasmid w/o the need of a promoter

G-less cassette

1 - a transcription unit after the TATA box, 233 base pairs long with no Gs in it; 2 - to terminate transcription, they put a row of Gs at the end; 3 - transcribe the DNA in the absence of cytosine, so transcription stops when you get to the row of Gs at the end; 4 - the row of Gs is used as a terminator; 5 - as a result, you get a very defined band

Formation of the pre-initiation complex

1 - TFIID binds to the promoter, recognizing the TATA box and the initiation site; 2 - TFIIA and TFIIB then bind near the TATA box; 3 - RNA polymerase and TFIIF then bind to the DNA (the polymerase is near the +1 site)

Binding of TFIIE and TFIIH

TFIIH uses ATP to unwind and open the DNA

Release of the polymerase from the promoter

in addition to abortive transcription, escape from the promoter is correlated with phosphorylation of the carboxy terminal domain of Pol II

TBP

1 - TATA binding protein; 2 - monomeric protein (single polypeptide); 3 - highly symmetrical; 4 - conserved between species (80% identical between yeast and humans in the conserved domain); 5 - TBP uses B-sheet to recognize the minor groove

TFIIA

1 - associates with TBP to prevent inhibitor binding; 2 - its not required for basal transcription; 3 - its reqruied for activation in some systems; 4 - it may prevent the binding of inhibitors to TBP

Experiment showign TFIIH has helicase activity

1 - denature very long DNA molecule; 2 - hybridize it to short, P32 labeled complementary DNA to it; 3 - when visualized on a gel, it wouldnt move far because of its size; 4 - after incubating the DNA w/ TFIIH, they could show release of the short complementary DNA from the very long DNA molecule in the gel

Steps in transcription initiation

1 - binding of general transcription factors and RNA polymerase II to promoter (closed complex); 2 - unwinding of DNA by TFIIH(open complex); 3 - phosphorylation of carboxyl terminus of RNA polymerase II by TFIIH and abortive initiation; 4 - release from promoter and most general transcription factors (promoter escape)

Elongation and processing of primary transcript

1 - release of initiaation factors is follwed by binding of elongation factors and processing of primary transcript; 2 - elongation, RNA processing and termination of transcription are all interconnected

Modification of RNA polymerase during transcription initiation

1 - the carboxy terminal domain (CTD) contains 7 amino acids (Y, S, P, T, S, P, S) that are repeated multiple times; 2 - Serine is modified by phosphorylation; 3 - CTD requried for processing mRNA (binds processing factors) and release of the initiation complex

Phosphorylation at different serine sites

phosphorylation at different sites is correlated to different stages of transcription; 1 - no phosphorylation during initiation complex formation (preinitiation); 2 - the first phosphorylation happens at serine5 and recruits capping enzymes, this is associated with the promoter escape stage; 3 - the second phosphorylation happens at serine2 and recruits splicing enzymes, this is associated with the elongation stage

Showing that TFIIH was responsible for phosphorylating Pol II

1 - Gel shifts were used to demonstrate TFIIH was responsible for phosphorylating Pol II; 2 - when you have TFIIH in the presence of ATP, you get a larger complex (phosphorylated complex) than you get w/o either component

5' cap

1 - all eukaryotic mRNAs have 5' "cap" (stabilizes RNA); 2 - first step is the removal of the gamma phosphate from the 5' end; 3 - then its the addition of GMP in unusual 5'-5' linkange (Bphosphate attacks aphosphate, releasing pp, forming triphosphate); 4 - then methylation of the position 7 on the G occurs (of the OH); 5 - RNAs are usually capped when tehy are 20-40 nucleotides long; 6 - after capping, the ser5 is dephosphorylated and capping enzymes dissociate

Termination of transcription in eukaryotes and polyA addition

termination of transcription in eukaryotes is closely linked to polyA addition; 1 - CstF and CPSF bind to P-CTD; 2 - transcription of polyA sequence causes transfer of factors to RNA; 3 - the RNA is cleaved and CstF is released; 4 - PAP (poly-A polymerase) is recruited and binds to teh 3' end; 5 - approximately 200 As are added to RNa (no template); 6 - polyA binding proteins then bind to A residues;

Torpedo model of transcription termination

1 - after the RNA is cleaved, Rat1 binds to the uncapped end still bound to the polymerase; 2 - it 5'->3' attacks the uncapped end and rapidly degrades RNA, destabilizing RNA polymerase, causing release of the factor and of the polymerase

Function of 5' cap and poly-A tail

1 - translation initiation (5' cap is bound by initiation factors and ribosome scans for first AUG); 2 - transport out of nucleus; 3 - stability of the mRNA; 4 - molecular biologists use poly-A tail to purify mRNA away from rRNAs and tRNAs which are not polyadenylated

Prokaryotes vs eukaryotes genes

1 - the genes in prokarytes are spaced very closely together; 2 - they can be cotranscribed into a single polycistronic mRNA; 3 - in eukaryotes the genes are farther away and are transcribed individually; 4 - the genes contain exons (the splicing of exons always occurs in order) and introns

RNA processing in eukaryotes

5' cap, 3' polyA addition, splicing

Leading up to RNA processing

1 - early experiments indicated that RNA was synthesized as a longer precursor; 2 - this larger class of RNAs was refereed to as heteronuclear RNA or hnRNA (many different sizes); 3 - the mRNA found associated with polyribosomes was an average much smaller than the hnRNA

Pulse-chase experiment

pulse: 1 - label RNAs with P32 for 30min; 2 - separate RNA by rate-zonal centrifugation (allows RNA to migrate according to size); 3 - most of the radioactivity (newly synthesized RNA) is larger than the total RNA (old and new) chase: 1 - stop transcription with actinomyocin D; 2 - allow cells to grow 3 hours; 3 - separate by rate-zonal centrifugation; 4 - the majority of the radioactivity is at the same size or smaller than the total RNA

Conclusions of hnRNA experiments

1 - mRNA is derieved from hnRNA: 2 - this process results in an overall decrease in size of RNA; 3 - other experiments showed that the size difference between hnRNA and mRNA is primarily due to removal of introns

Splicing

1 - the splicing reaction is catalyzed by enzymes composed of a complex of RNA and protein referred to as snRNPs (small nuclear ribonucleoprotein particles); 2 - the RNA components are abunant, uridine-rich RNAs and are referred to as U1, U2, U4/U6 and U5; 3 - each snRNA is associated with 10 or more proteins

Overview of splicing

1 - assembly of spliceosome: 5 snRNPs (small nuclear ribonucleoprotein particles - U1, U2, U4, U5, U6) recognize the splice sites and interact/bind with each other (spliceosome) to bring the sites together; 2 - 2 transesterification steps: brach-5' splice site (formation of lariat), 5' splice site-3' splice site, release of intron as lariat

Spliceosome formation

1 - first step is the recognition of the junction (conserved sequences) between the exons and introns; 2 - recognition involves RNA-RNA base pairing assisted by proteins; 3 - four conserved sequences have been found to be necessary (5' splice site, branch point (generates lariat), pyrimidine track, 3' splice site)

5' splice site

1 - near the exon that is closer to the 5' end of the mRNA; 2 - donor site

3' splice site

1 - near the exon that is closer to teh 3' end of the mRNA; 2 - acceptor site

Branch site

1 - closer to the 3' end; 2 - not as conserved

Formation of spliceosome (specific)

1 - binding of U1 snRNP to 5' splice site, BBP to branch point and U2AF to poly pyrimidine; 2 - release of BBP and binding of U2 to branch point; 3 - binding of U4/U6,U5 complex and release of U2AF

RNA recognition during spliceseome formation

recognition involves U1 RNA base pairing to 5' splice site and U2 RNA base pairing to branch point (the A on the branch point is not basepaired, its bulging out)

Spliceosome rearrangement

1 - a major rearrangement (U6 basepairs with U2 instead of U4) in the spliceosome proceeds or is concurrent with teh transesterification reactions; 2 - this rearrangement places U6 in position to catalyze the reactions

Spliceosome rearrangement and transesterification reactions

1 - rearrangement releases U1 from 5' splice site and U4 from U6; 2 - U6 then catalyzes first transesterification reaction; 3 - U5 assists in the second transesterification reaction; 4 - U2, U5, U6 are the only snRNPs left during catalysis

First transesterification reaction

1 - involves the nucleophilic attack by the 2'OH on the brach point A on the 5' splice site; 2 - this intermediate is referred to as a lariat, the branch poitn A has 3 phosphodiester bonds

Second transesterification reaction

1 - involves the nucleophilic attack by the 3'OH at teh 5' splice site on teh phosphodiester bond at teh junction of the 3' splice site; 2 - the intron is released as a lariat, the spliceosome dissociates and teh mRNA (spliced exons) is ready to be transported out of the nucleus

Alternative splicing

1 - constitutive (two or more splice variants are always made); 2 - regulated (splice variants are made in only certain cell types or certain times of development); 3 - you can remove/skip exons, leave a little bit of the intron in the mRNA, retain whole introns; 4 - this is humans are so complex compared to other organisms w/ similar number of genes

Advantage of having introns that need to be eliminated before mRNA can be translated

allowed evolution to proceed at an increased pace (1 - alternative splicing allows a variety of related proteins to be synthesized from a single gene; 2 - exons frequently encode different domains of a protein which can be combined via DNA rearrangements to generate new proteins relatively quickly (exon shuffling))

Exon shuffling (evolution)

1 - because of repeated DNA sequences (transposable elements, etc), recombination of exons (the exons cross over between the strands) occurs when 2 strands w/ homologous sequences; 2 - you end up with 2 new genes (each of which still has its splice sites)

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