Eukaryotic Transcription

Transcription

1 Eukaryotic Transcription I. Differences between eukaryotic versus prokaryotic transcription. II. (core vs holoenzyme): RNA polymerase II - Promotor elements. - General Pol II transcription factors (GTF). - TBP and TAFs. - Mediator / SRB complex. - Swi/Snf and SAGA complex. III. Regulation of RNA pol II transcription. - Transcription initiation (yeast Gal4 system) - Activators, repressor, enhancer and silencer. - Transcription elongation (HIV Tat-tar system). IV: Chromatin remodeling during transcription. - Role of Swi/Snf complex and TAFs.

2 I. Eukaryotes vs prokaryotes transcription. Eukaryotes Most mrnas are monocistronic (generally one gene for one protein). Transcription and translation of most eukaryotes are spatially separated. Multiple RNA polyermases. Transcription factor directly binds to the DNA template, but not to the RNA pol. Requires ATP for initiation. Nucleosome / chromatin. Prokaryotes Polycistronic mrna. Coupling transcription and translation. Single RNA polyermase RNA pol holoenzyme itself, by associated sigma factor. No ATP Naked DNA I. Eukaryotes vs prokaryotes transcription.

3 RNA polymerase II is capable of transcribing DNA templates, but it will not begin transcription at the correct site. Hence it is competent for elongation but not initiation.?????? Hypothesis: There must be a cis-acting DNA sequence and trans-acting factor that recruits or direct RNA Pol II at the site of transcription initiation. enhancer upstream regulatory elements Start of Transcription core (-200) (-45) (+40) core promoter Core Promoter : Minimal region required for basal transcription. Regulatory Elements: Upstream regulatory sequence (Proximal element) - usually within a few hundred bases of the promoter. Enhancer sequence (Distal element) that functions at a distance (even downstream or within the transcription unit) and orientation independent manner.

4 Identifying regulatory elements: - Systematically mutate DNA and connect to a convenient reporter gene. luciferase chloramphenical acetyl transferase (CAT) ß-galactosidase. - Transfect DNA back into a suitable eucaryotic cell. - Measure reporter gene activity. Caveats: Cell lines are usually immortal so they carry mutations in cell cycle control. Response may be cell-type specific - specific factors or combination of factors may be absent. Chromatin structure may be abnormal. If DNA has randomly inserted into the genome, neighboring sequences can affect transcription. enhancer upstream regulatory elements Start of Transcription core (-200) (-45) (+40) core promoter Start of Transcription (-37 to -32) (-31 to -26) (-2 to +4) (+28 to +32) BRE TATTA Int DPE They are essential for the assembly of the general transcription factors and Pol II complex and the initiation of transcription, and therefore are required for directing where transcription starts. The Inr element encompasses the transcription start site. Found in both TATA-containing as well as TATA-less core promoters. DPE is found most commonly in TATA-less promoters. TFIIB is able to bind directly to the BRE in a sequence-specific manner.

5 enhancer upstream regulatory elements Start of Transcription core (-200) (-45) (+40) core promoter Start of Transcription (-37 to -32) (-31 to -26) (-2 to +4) (+28 to +32) BRE TATTA Int DPE In promoters with TATA box -initiation occurs ~35 bp downstream. Often more than one start site Start site variability greater in S. cerevisiae (40-100bp)

6 enhancer upstream regulatory elements Start of Transcription core (-200) (-45) (+40) Proximal element: (upstream regulatory sequence ): core promoter Relatively GC-rich. Allows binding of Sp1 and related transcription factors (activator or repressor. Often present as multiple copies and can act synergistically. Upstream regulatory sequence (Proximal element):

7 enhancer upstream regulatory elements Start of Transcription core (-200) (-45) (+40) core promoter Distal element: (enhancer / silencer): Facilitate assembly of pre-initiation complex, directly or indirectly. Enhancer: function as binding sites for specific transcription activator (often found in developmentally regulated genes). Function in either orientation at variable distance (0.5 kb - 10 kb). In mammalian system can be positioned downstream of TATA. Silencer also binds to distal element and function as transcriptional repressor. Three distinct DNA-dependent RNA polymerases in the nucleus: RNA pol I, II, and III. Two additional RNA polymerases in organelles: one in mitochondria and one in chloroplasts. Three nuclear RNA Pols were first identified as distinct polymerases because they elute from columns at different salt concentrations and can be classified by the sensitivity to alpha-aminitin (chemical inhibitor of Pol II transcripton).

8

9 Pol I Approx % of total RNA synthesized the precursors for the 28S, 18S, and 5.8S ribosomal RNAs. Regulation is minimal and synthesize stable RNA. Pol II (10-15% of total) typically synthesized mrnas that are translated into proteins. Also some snrnp RNAs involved in mrna splicing. Regulation is diverse and transcripts are less stable. Pol III (5-10% of total) synthesize small RNAs, such as trnas, 5S RNA, some of the other spliceosome RNAs, such as U6 and H1. Regulation is minimal and synthesize stable RNA. None of the subunits appreas to be related to the bacterial sigma-subunit

10 Bushnell, Cramer, Kornberg, Proc Natl Acad Sci USA 99 (2002) p.1218

11 Tandem repeat heptad sequence: ( Tyr Ser Pro Thr Ser Pro Ser ) n Mouse (n = 52) Budding / Fission yeast (n = 26 / 29) Plasmodium (n = 15)

12 CTD of RNA polymerase II Two forms of RNA Pol II: Pol II A: not phosphorylated and preferentially enters the pre-initiation complex. pol IIO: physophorylated at Ser residue and is found in elongating complex. Conversion of IIA to IIO occurs shortly after the transition from initiation to elongation (promoter clearance. RNA pol II lacking the CTD is able to initate transcription in vitro but not from TATA-less promoter. Hypothesis: There must be a cis-acting DNA sequence and trans-acting factor that recruits or direct RNA Pol II at the site of transcription initiation. Approach:??? Biochemically isolate factors that are required for promotor specific transcription (mammalian and viral system). Genetically identify mutants and suppressor genes involved in transcription (yeast and flys).

13 Assay system to isolate transcription factor biochemically. Assay system to isolate transcription factor. G-less Cassette p(c2at) AdML G-less Cassette pml(c2at) - DNA template - RNA polymerase (purified separately) - NTP (ATP, UTP, 32P-CTP and GTP*) - TFIIs (nuclear extract or column fractions) (1) p(c2at) - GTP (2) pml(c2at) - GTP (3) p(c2at) (4) pml(c2at) (5) p(c2at) + RNaseT1 + 2 O-mGTP (6) pml(c2at) + RNaseT1 + 2 O-mGTP Accurate and specific transcription from promoter containing template. Specific transcription can be quantitate by direct measurement of total RNA synthesis.

14 Assay system to isolate transcription factor. Assembly of GTF at the promoter in vitro: Gel mobility shift assay.

15 Assembly of GTF at the promoter in vitro: Gel mobility shift assay. TFIIA Assembly of GTF at the promoter in vitro: Gel mobility shift assay. Purified RNA Pol II, TFII D, A, B, or E do not bind stably to promoter when added individually (lane 1-5) When all the individual components were added (RNA Pol II, TFII D, A, B, ande)formed6complex(lane6) TFIIA is not necessary, but TFIID is absolutely required for complex formation (lane 7, 8 & 12). Lack of TFIIB or RNA Pol II abolished formation of slow migrating complex (lane 9 & 10). This suggest that TFIIB may involved in recruitment of pol II. Note that addition of TFIIB can form complex3intheabsenceofpolii. Omission of TFIIE prevented formation of complex 6 & 7 (compare lanes and lane 6-8).

16 Assembly of GTF at the promoter in vitro:dnase 1 Footprint. Coding Strand Non-Coding Strand Complex2:TFIIA,-D. - Protection at -42 to Complex3:TFIIA,-D,-B. - Similar to complex 2 pattern with additional protection -10 to +10 region. Complex 4 and 5: TFFIIA, -D, -B, pol II. - Same upstream boundary (-42) but cleavage downstream was strongly suppressed (up to +20). Complex 6 and 7: TFIIA, -D, -B, pol II and - E. - Enhance cleavage downstream of the promoter (to about +30 on coding strand). (1 subunit) TFIIE TFIIF TFIIH TBP (2 subunits) (9 subunits) (1 subunit) (2 subunits) TAFs (12 subunits)

17 TFIIE: TFIIEiscomposedtof2subunits(56and 34 kda). TFIIF binds prior to TFIIE (lane 4). Requires both TFIIE subunit to form a complex (lane 5-8) - negative result!. Recombinant TFIIE was capable of forming a complex similar to purified TFIIE (lane 4 and 10). Omission of TFIID prevented formation of complex. TFIIH The only GTF with known enzymatic activity: DNA-dependent ATPase ATP-dependent helicase CTD kinase MostcomplexGTFwithatotalmassof 500 kda (~ size of RNA pol II). Converts RNA pol IIA to pol II O (phosphorylation) at CTD Ser 5. TFIIH alters the mobility of the PIC complex in the presence of ATP

18 TFIIH (helicase / ATPase activity appears to act at the promoter clearance step, a transition stage between initiation and elongation. General Transcription Factor Factor Subunits Function TFIID TBP 1 Core promoter recognition (TATA); Recruit TFIIB. TAFs 12 Core promoter recognition (non-tata); positive and negative regulatory function. TFIIA 3 Stabilize TBP binding: stabilization of TAF- DNA interaction; anti-repressor functions. TFIIB 1 pol II-TFIIF recruitment; start-site selection by pol II. TFIIF(w/Pol II) 2 Promoter targeting of pol II; destabilization of non-specific pol II-DNA interactions. TFIIE 2 TFIIH recruitment; modulation of TFIIH helicase, ATPase and kinase activity; enhancement of promoter melting(?). TFIIH 9 Promoter clearance by CTD kinase activity. Promoter melting by helicase activity(?).

19 TATA binding protein: TBP : TATA binding protein. The C-terminal 180 amino acid of TBP is the TATA-binding domain. TBP binds and grossly distorts the minor groove of TATA box