Biochemistry 201 Biological Regulatory Mechanisms January 26 2015

Biochemistry 201 Biological Regulatory Mechanisms January 26, 2015 C. Gross, Lecture 3 Eukaryotic transcriptional regulation References A few of the many insights from RNA polymerase structures Cramer, P. (2002) Multisubunit RNA polymerases. Curr Opin Struct Biol 12: 89 -97. Murakami KS, Darst SA. (2003) Bacterial RNA polymerases: the holo story. Curr Opin Struct Biol 13: 31 -9. *Cramer, P. (2004) RNA polymerase II structure: from core to functional complexes. Curr Opin Genet Dev 14: 218 -26. Review. Wang, D. Bushnell DA, Westover KD, Kaplan, CD, Kornberg RD. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell. 2006 Dec 1; 127(5): 941 -54. Kostrewa D, Zeller ME, Armache KJ, Seizl M, Leike K, Thomm M, Cramer P. (2009) RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature. 462: 323 -30. Chromosome conformation capture (CCC) de Wit, E. and de Laat, W. (2012) A decade of 3 C technologies: insights into nuclear organization. Genes Dev. 26: 11 -24. Mediator and Other Components *Kornberg, R. D. (2005) Mediator and the mechanism of transcriptional activation. Trends in Biochemical Sciences 30: 235 -239. Fan, X, Chou, DM, & Struhl, K. (2006). Activator-specific recruitment of Mediator in vivo. Nature Structural & Molecular Biology, 13(2), 117 -20. Sikorski TW and Buratowski. (2009). The basal initiation machinery: Beyond the general transcription factors. Current Opinion in Cell Biology. 21 344 -351.

What do activators do? Cosma, MP, Tanaka, T, & Nasmyth, K. (1999). Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle- and developmentally regulated promoter. Cell, 97(3), 299 -311. Bryant, GO, & Ptashne, M. (2003). Independent recruitment in vivo by Gal 4 of two complexes required for transcription. Molecular Cell, 11(5), 1301 -9. Bhaumik, S. R. , Raha, T. Aiello, D. P. , and Green, M. R. (2004) In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer. Genes Dev 18: 333 -343. Vakoc, CR, Letting, DL, Gheldof, . . . Blobel, GA (2005) Proximity among Distant Regulatory Elements at the B–Globin Locus Requires GATA-1 and FOG-1. Molecular Cell 17: 453 -462 Fishburn, J. , Mohibullah, N. and Hahn, S. (2005) Function of a eukaryotic transcription activator during the transcription cycle. Molecular Cell 18: 369 -378. Bulger M and Groudine M. Functional and Mechanistic Diversity of Distal Transcription Enhancers (2011). Cell 144: 327 -39 Role of the RNA Pol II CTD *Mc. Cracken, S, Fong, N, Yankulov, K, et al. (1997). The C-terminal domain of RNA polymerase II couples m. RNA processing to transcription. Nature, 385(6614), 357 -61. Tietjen, J. ……Ansari, A. Chemical-genomic dissection of the CTD code (2010) NMSB: 17: 1154 -1162 Mayer, A. …. Cramer, P. Uniform transitions of the general Pol II transcription apparatus (2010) NMSB 17: 1272 -79 Buratowski, S (2009) progression through the RNA polymerase II CTD cycle ( Review). Mol Cell 36: 541 -546 Chapman, R… Eick, D. Molecular evolution of the RNA polymerase CTD. Trends in Genetics (2008): Jun; 24(6): 289 -96. Epub 2008 May 9. Review. PMID: 18472177 Genome wide elongation technologies Churchman LS, Weissman JS. (2011) Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469: 368 -73. doi: 10. 1038/nature 09652. (Net-seq) Kwak, H… and Lis, JT (2013) Precise maps of RNAP reveal how promoters direct initiation and pausing. Science 339: 950 (PRO-seq)

Elongation Control BBA 2013 -- Issue 1874 devoted to reviews of transcription elongation Yamaguchi, Shibata and Handa (2013): Transcriptionelongation factors DSIF and NELF: Promoter proximal Pausing and Beyond. BBA 1829: 98 -104 Note: this issue of BBA has many elongation reviews; most not too good. Kwak and Lis (2013) Control of transcriptional Elongation Annual review Genetics 47: 483 -508 Jonkers, Kwak and Lis (2014) Genome-wide dynamics of Pol. II elongation and its interplay with promoter proximal pausing, chromatin and exons. E-life: 2014; 3: e 02407. DOI: 10. 7554/e. Life. 02407 Veloso, Kirkconnell, magnuson, Biewen, Paulsen, Wilson and Mats Ljungman. (2014) Rate of elongation by RNA polymerase II is associated with specific gene features and epigenetic modifications. Genome research 24: 896– 905 Core, Waterfall and Lis (2008) Nascent RNA Sequencing Reveals widespread pausing and divergent initiation at Human promoters. Science 322: 1845 -1848 Zhou Q, Li T, Price DH (2012) RNA polymerase II elongation control. Annu Rev Biochem. 2012; 81: 119 -43. Rougvie A and Lis JT (1988) The RNA Polymerase II Molecule at the 5’ end of the uninduced hsp 70 gene of D. melangaster is transcriptionally engaged. Cell 54: 795 -804 Zobeck, KL…. Lis Jt (2010) Recruitment timing and dynamics of transcription factors at the Hsp 70 Loci in Living Cells Mol Cell 40 965 -75 Nechaev S…. . Adelman K. (2010) Global Analysis of short RNAs reveals widespread Promoter Proximal Stalling and Arrest of Pol II in Drosophila Science 327: 335 -38 Gilchrist, DA, ……Adelman, K. (2010) Pausing of RNA Polymerase II Disrupts DNA specified Nucleosome Organization to enable precise gene regulation. Cell 143: 540 -51 Chen, Y. , …. Handa, H. (2009) DSIF, the Paf 1 complex and Tat-SF 1 have nonredundant, cooperative roles in RNA polymerase II elongation. Genees Dev 23: 2765 -77 Liu, Y……Hahn, S. (2009) Phosphorylation of the transcription elongation factor Spt 5 by yeast Bur 1 kinase stimulates recruitment of the PAF complex. MCB 29: 4852 -63 Wu, C-H, …. Gilmour, D. ( 2003)NELF and DSIF cause promoter proximal pausing on the Hsp 70 promoter in drosophila. Genes Dev 17: 1402 -14

RNA polymerase elongation rate and splicing Braberg, Jin, Moehle, Chan, Wang, Shales……Guthrie, Kaplan Krogan ( 2013) From Structure to Systems: High resolution Quantitative genetic Analysis of RNA polymerase II. Cell 154, 775 -788 Fong, Kim, Zhou, Jim Qiu, Saldi Diesner, Jones, Fu and Bentley (2014) Ore 0 m. RNA splicing is facilitated by an optimal RNA polymerase II elongation rate Genes and Dev 28: 2663 -2676

Important Points 1. DNA looping allows proteins bound to distant sites on DNA to interact. 2. Transcription initiation at Pol II promoters on naked DNA templates in vitro requires the general transcription factors in addition to RNA polymerase II. 3. . In vivo, transcription initiation also requires activators – proteins that bind directly to enhancers – as well as Mediator and enzymes that modify chromatin structure. 4. At a typical eukaryotic promoter, activators guide the assembly of Mediator, the general transcription factors, RNA polymerase and chromatin-modifying enzymes, often through weak, relatively non-specific interactions. There appears to be no set order of assembly from one promoter to the next. Moreover, different promoters have different requirements for these components. 5. The RNA polymerase CTD is a long series of 7 -amino acid repeats. When transcription is initiated, serine 5 of the repeat is phosphorylated by TFIIH. As elongation proceeds, serine 5 is gradually dephosphorylated and serine 2 is gradually phosphorylated by enzymes carried along with the RNA polymerase. This dynamic pattern of modification couples transcription to processing of the newlysynthesized RNA. 6. Promoter proximal pauses ( 20 -50 nucleotides) are extensively used for regulation in eukaryotes. 7. Transcription takes place in a chromatin world and they exhibit a 2 -way interaction

New challenges for transcription in eukaryotic cells 1. Three polymerases 2. Much more complex pattern of gene expression Proliferation of trx factors Regulation at a distance ( enhancers) Combinatorial control 3. Transcription takes place in a chromatin world Constant 2 -way interplay between trx and chromatin 4. Complex processing of m. RNA Pol. II CTD serves as a platform for coordinating processes

Pol. II initiation factors and core promoter

Purification scheme for partially purified general transcription factors. Fractionation of He. La nuclear extract (Panel A) and nuclear pellet (Panel B) by column chromatography and the molar concentrations of KCl used for elutions are indicated in the flow chart, except for the Phenyl Superose column where the molar concentrations of ammonium sulfate are shown. A thick horizontal (Panel A) or vertical (Panel B) line indicates that step elutions are used for protein fractionation, whereas a slant line represents a linear gradient used for fractionation. The purification scheme for pol II, starting from sonication of the nuclear pellet, followed by ammonium sulfate (AS) precipitation is shown in Panel B. (Figures are adapted from Flores et al. , 1992 and from Ge et al. , 1996) NAME # OF SUBUNITS FUNCTION TFIIA 3 Antirepressor; stabilizes TBP-TATA complex; coactivator TFIIB 1 Recognizes BRE; Start site selection; stabilize TBP-TATA; pol II/TFIIF recruitment TFIID TBP TAFs 1 ~10 Binds TATA box; higher eukaryotes have multiple TBPs Recognizes additional DNA sequences; Regulates TBP binding; Coactivator; Ubiquitin-activating/conjugating activity; Histone acetyltransferase; multiple TAFs TFIIF 2 Binds pol II; facilitates pol II promoter recruitment and escape; Recruits TFIIE and TFIIH; enhances efficiency of pol II elongation TFIIE TFIIH 2 9 Recruits TFIIH; Facilitates forming initiation-competent pol II; promoter clearance ATPase/kinase activity. Helicase: unwinds DNA at transcription startsite; kinase phosphorylates ser 5 of RNA polymerase CTD; helps release RNAP from promoter

The Pol II promoter has many recognition regions Positions of various DNA elements relative to the transcription start site (indicated by the arrow above the DNA). These elements are: BRE (TFIIB recognition element); there is also a second BRE site downstream of TATA (TATA Box); Inr (initiator element); DPE (downstream promoter element); DCE (downstream core element). MTE (motif ten element; not shown) is located just upstream of the DPE.

The first two steps of Eukaryotic transcription On a supercoiled naked DNA template and in archae, TBP and TFB are sufficient formation of the pre-initiation complex (PIC), suggesting that they are key to the mechanism of transcription initiation in eukaryotes TFB TBP Promoter Many archae have a proliferation of TBPs and TFBs, suggesting that they provide choice in promoters, akin to alternative s.

Transcription Initiation by RNA Pol II The stepwise assembly of the Pol II preinitiation complex is shown here. Once assembled at the promoter, Pol II leaves the preinitiation complex upon addition of the nucleotide precursors required for RNA synthesis and after phosphorylation of serine resides within the enzyme’s “tail”. PIC = preinitiation complex

The GTF’s are not enough—what else is needed? The concept of a co-activator

The GTFs are not sufficient to mediate activation: Discovery and isolation of Mediator from Yeast GTFs and RNA Pol II Tx 1 unit VP 16 GAL 4 1 unit crude lysate 10 units 4 years mediator 50 units

Is Mediator Required for Transcription of all Pol II -transcribed genes? Control: ts subunit of Pol II Rpb 1 high T Rpb 1 ts Compare levels of all m. RNAs using microarrays (37˚C for 45 min) m. RNAs decrease over time according to their half-lives. A few m. RNAs remain at some level (stable m. RNAs). Subunit of Mediator Srb 4 ts Same basic pattern Subunit of TF II H Kin 28 ts Same basic pattern

Mediator is very large and has diverse roles A) PIC model from EM-study of pol. II (brown)-TFIIF (light blue) and X-ray structure of TBP (white) -IIB (yellow)--DNA; Arrow indicates direction of trx B) Model for PIC-mediator was produced by superimposing an EM structure of Mediator-Pol. II on the PIC in A; head, middle and tail regions shown The Head region interacts with Pol. II-TFIIF complex; the mutants with general effects on Trx are located in this region; the tail region interacts with activators; mutants have more specific effects on transcription

SAGA is another important complex with multiple roles in transcription, including being a coactivator The core of SAGA, containing the Taf substructure (Yellow), is surrounded by three domains responsible for distinct functions: activator binding (Tra-1), histone acetylation Gcn 5), and TBP regulation (Spt 3). This structural organization illustrates an underlying principle of modularity that may be extended to our understanding of other multifunctional transcription complexes.

The TAFs in TFIID also serve as coactivators

Assembly of PIC in presence of mediator, activators and chromatin remodelers

Many Paths to the PIC Buratowski, 2009 The factors and assembly pathways used to form transcriptionally competent preinitiation complexes can be promoter dependent. (1) TBP assembling onto promoter regions via TFIID leads to recruitment of the other basal initiation factors, as outlined in the stepwise assembly pathway. In S. cerevisiae, this pathway is most often utilized at TATA-less genes. At some mammalian promoters, histone H 3 K 4 trimethylation helps to recruit the TFIID complex. (2) Mediator bridges interactions between activators and the basal initiation machinery, and can stimulate basal transcription as well. At some promoters Mediator can recruit TFIIH and TFIIE independently of RNApo. II. (3) TBP can also be brought to promoters by the SAGA complex. In S. cerevisiae, this pathway is most utilized at TATA containing promoters. The Mot 1 and NC 2 complexes can repress this pathway by actively removing TBP from the TATA element. (4) Mot 1 and NC 2 can also have a positive role in transcription by removing non-productive TBP complexes from DNA, thereby allowing functional PICs to form.

Genomic Level Snapshots of transcription factor binding sites Ch. IP-Ch. IP-seq Ch. IP-exo Bioinformatic approaches Phylogenetic footprinting

Regulatory sequences expand in number and complexity with increased complexity of the organism ~ 30 -100 bp ~ 100 s bp Could be 50 k. B or more Are distant enhancers in proximity to the promoter? Chromosome conformation capture-3 C

3 C 5 C

1 st example of 3 C applied to enhancers: b globin locus: 200 k. B A hypothetical model of the active chromatin hub (ACH) is shown to illustrate the 3 D nature of the ACH (not to scale), not the actual position of the elements relative to each other in vivo. Red indicates the active regions (hypersensitive sites and active genes) of the locus forming a hub of hyperaccessible chromatin (ACH). The Inactive regions of the locus, having a more compact chromatin structure are indicated in gray, with the inactive h 1 and y genes in lighter gray. Molecular Cell, Vol. 10, 1453– 1465, December, 2002

Elongation control in eukaryotes

RNA polymerase II CTD Heptad repeat unit YSPTSPS P P P 2 5 7 Plasmodium: 5 Yeast: 26 Mammals: 52 CTD is located adjacent to RNA exit channel Palangat M et al. PNAS 2005; 102: 15036 -15041 © 2005 by National Academy of Sciences

Phosphorylation state of Pol. I CTD during transcription 2 5 7 YSPTSPS Stage of transcription Initiation Kinase/phosphatase YSPTSPS (Unphosphorylated) TF II H, Mediator Transition to elongation YSPTSPS (Ser 5) P p. TEFb/Cdk 9 Elongation (Ser 2, 5) Further elongation (Ser 2) Termination (Unphosphorylated) In S. cerevisiae: Cdk 1 and Bur 1 YSPTSPS P P Phosphatases (Rtr 1(2? ) YSPTSPS P YSPTSPS Phosphatases (Fcp 1, ssu 72)

A new step in transcription in higher eukaryotes Prokaryotes NTPs KB R+P RPc Kf RPo Initiation Abortive Initiation transition Elongating Complex Elongation/termination Higher Eukaryotes: same through abortive initiation, but new step before elongation PIC ( does abortive initiation) Promoter-proximal pause: +20 - +50 NELF: Negative elongation factor; DSIF: DRB (5, 6 -dichloro-1 -beta-D-ribofuranosyl-benzimidazole) sensitivity-inducing factor-Spt 4+5 Potential roles for paused polymerase: antagonize nucleosome formation to keep promoter open; synchronous activation; checkpoint

What is the major role of the Pol II CTD? Mouse RNA Pol II wt 52 - ama. R 5 50 hrs. He. La cells Introduce CTD construct CTD Splicing, processing of 3’ examine end, termination RNAs were all affected - amanitin Nature 385: 357 (1997)

Specific Processes are connected to each Phosphorylated Form of the CTD Status Transcription RNA-Processing Chromatin Unphosphorylated Activation (mediator) Serine 5 P early termination m. RNA capping H 3 K 4 modification (Sc. N 4 E 1 complex) (capping enzyme) Set 1 complex progression to elongation Nucleosome mobility (Cdk 9 kinase via capping Cdk 9/bur 1 for Spt 5 enzyme); Bur 1 kinase) Serine 2 P/5 P H 3 K 36 methylation (Set 2 ) Serine 2 P late termination polyadenylation histone chaperone (Rtt 103) (Pcf 11) Spt 6

Histone marks: transcriptional regulators or by-products? Approach: track residue specific modifications in living cells in a strain with a GFP-glucocorticoid receptor and an inducible tandem array Kimura doi: 10. 1038/nature 13714 Results: 1. Preacetylated prior to addition of glucocorticoid 2. High acetylation correlated with rapid gene activation and rapid transition to elongation

TECs are community organizers The major steps in m. RNA processing (trx, 5’ capping, poly. A addition, splicing) all occur together on a transcript extruded from the exit channel of RNAP although they can be reconstituted independently in vivo Principles of “cotranscriptionality” to integrate nuclear metabolism 1. Permits coupling between different biogenesis steps; eg crosstalk; suspected when decreasing one step has effects on 2 nd; could always be indirect a. Landing pad—increase concentration of reactants—proteins involved in capping etc b. Allosteary: guanosyl transferase of capping enzyme activated by interaction with phosphorylated CTD c. Kinetic coupling—optimize timing 2. Impose order or control a. Juxtaposition of proteins permits assembly, competitive interactions handoffs; often mutually exclusive PPis b. directions emanating from phopshorylation state of CTD 3. A locator for nuclear machines –DNA repair, modification etc Bentley: Cotranscriptionality Mol cell rev 2009

Kinetic coupling model for splicing Approach: Measure splicing efficiency in a series of slow and fast RNAP; Importantly double slow + fast returned splicing to normal Caveats: not true for all splice sites; some reverse, some unaffected