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Transcription and Its Regulation January 21 –Mechanism of Transcription Initiation

Transcription and Its Regulation January 21 –Mechanism of Transcription Initiation January 23– Regulation of of Transcription Initiation January 27–Mechanism and regulation of Transcription Elongation January 30– In class discussion of problem set

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Transcription and Its Regulation January 21 –Mechanism of Transcription Initiation

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  1. Transcription and Its Regulation January 21 –Mechanism of Transcription Initiation January 23– Regulation of of Transcription Initiation January 27–Mechanism and regulation of Transcription Elongation January 30– In class discussion of problem set Mechanism of Transcription Initiation References I. General Chapter 12 of Molecular Biology of the Gene 6th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M, Losick, R. 377-414. 2. Reviews Murakami KS, Darst SA. (2003) Bacterial RNA polymerases: the wholo story. CurrOpinStructBiol 13:31-9. Campbell, E, Westblade, L, Darst, S., (2008) Regulation of bacterial RNA polymerase factor activity: a structural perspective. Current Opinion in Micro. 11:121-127 Herbert, KM, Greenleaf, WJ, Block, S. (2008) Single-Molecule studies of RNA polymerase: Motoring Along. Annu Rev Biochem. 77:149-76. Werner, Finn and Dina Grohmann (201). Evolution of multisubunit RNA polymerases in the three domains of life. Nature Rev. Microbiology 9: 85-98 Grunberg, S. and Steven Hahn (2013) Structural Insights into transcription initiation by RNA polymerase II. TIBS 38: 603-11. 3. Studies of Transcription Initiation Roy S, Lim HM, Liu M, Adhya S. (2004) Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step. EMBO J. 23:869-75. Sorenson MK, Darst SA. (2006).Disulfide cross-linking indicates that FlgM-bound and free sigma28 adopt similar conformations. Proc NatlAcadSci U S A. 103:16722-7.

  2. Young BA, Gruber TM, Gross CA. (2004) Minimal machinery of RNA polymerase holoenzyme sufficient for promoter melting. Science.303:1382-1384 *Kapanidis, AN, Margeat, E, Ho, SO,.Ebright, RH. (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science.314:1144-1147. Revyakin A, Liu C, Ebright RH, Strick TR (2006) Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science. 314: 1139-43. Murakami KS, Masuda S, Campbell EA, Muzzin O, Darst SA (2002). Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science. 296:1285-90. 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. Discussion Paper **Feklistov A and Darst, SA (2011) Structural basis for Promoter -10 Element recognition by the Bacterial RNA Polymerase s Subunit. Cell147: 1257 – 1269 Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock and Key to Transcription: s –DNA Interaction. Cell: 147: 1218-1219

  3. Reviews Articles: Chromosome conformation capture (CCC) technologies de Wit, E. and de Laat, W. (2012) A decade of 3C technologies: insights into nuclear organization. Genes Dev. 26: 11-24. Elongation BBA2013-- Issue 1874 devoted to reviews of transcription elongation General Transcription Factors Matsui, T., Segall, J., Weil, P.A., and Roeder, R.G. (1980) Multiple factors required for accurate initiation of transcription by purified RNA polymerase II. J BiolChem 255: 11992-11996. Thomas, M.C., & Chiang, C.M. (2006). The general transcription machinery and general cofactors. Critical reviews in Biochemistry & Molecular Biology, 41(3), 105-78. Muller, F, Demeny, MA, & Tora, L. (2007). New problems in RNA polymerase II transcription initiation: matching the diversity of core promoters with a variety of promoter recognition factors. The Journal of Biological Chemistry, 282(20), 14685-9. 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.

  4. Key Points • 1. Multisubunit RNA polymerases are conserved among all organisms • 2. RNA polymerases cannot initiate transcription on their own. In bacteria s70is required to initiate transcription at most promoters. Among other functions, it recognizes the key features of most bacterial promoters, the -10 and -35 sequences. • 2. E. coli RNA polymerase holoenzyme, (core + s) finds promoter sequences by sliding along DNA and by transfer from one DNA segment to another. This behavior greatly speeds up the search for specific DNA sequences in the cell and probably applies to all sequence-specific DNA-binding proteins. • 3. Transcription initiation proceeds through a series of structural changes in RNA polymerase,s70and DNA. • 4. A key intermediate in E. coli transcription initiation is the open complex, in which the RNA polymerase holoenzyme is bound at the promoter and ~12 bp of DNA are unwound at the transcription startpoint. Open complex formation does not require nucleoside triphosphates. Its presence can be monitored by a variety of biochemical and structural techniques. • 5. Recognition of the -10 element of the promoter DNA is coupled with strand separation • 6. When the open complex is given NTPs, it begins the ‘abortive initiation’ phase, in which RNA chains of 5-10 nucleotides are continually synthesized and released. • 7. Through a “DNA scrunching” mechanism the energy captured during synthesis of one of these short • transcripts eventually breaks the enzyme loose from its tight connection to the promoter DNA, and it begins • the elongation phase. • 7. Aspects of the mechanism of initiation are likely to be conserved in eukaryotic RNA polymerase

  5. transcription (RNA processing) snRNAs miRNAs rRNAs mRNAs Other non-coding RNAs (e.g. telomerase RNA) translation proteins Transcription is Important

  6. Transcription/Splicing/Translation Provide A Large Range of Protein Concentrations

  7. I. RNA polymerases

  8. Cellular RNA polymerases in all living organisms are evolutionary related SubunitsofRNAP LUCA-Last universal common ancestor Acommon structural and functional frame work of transcription in the three domains of life

  9. Structure of RNAP in the three domains Universally conserved Archaeal/eukaryotic Bacteria Archaea Eukarya Transcription Extra RNAP subunits provide interaction sites for transcription factors, DNA and RNA, and modulate diverse RNAP activities Werner and Grohmann (2011), Nature Rev Micro 9:85-98

  10. TYPE OF POLYMERASE GENES TRANSCRIBED RNA polymerase I 5.85, 18S, and 28S rRNA genes RNA polymerase II all protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes, and some snRNA genes RNA polymerase III tRNA genes, 5S rRNA genes, some snRNA genes and genes for other small RNAs The rRNAs are named according to their “S” values, which refer to their rate of sedimentation in an ultra-centrifuge. The larger the S value, the larger the rRNA. Eukaryotic Cells have three RNA polymerases

  11. Evolutionary relationships of general transcription factors s Initiation s Gre Transcript cleavage Elongation LUCA may have had elongating, not initiating RNA polymerase

  12. II. Challenges in initiating transcription • RNAP is specialized to ELONGATE, not INITIATE 2. Initiating RNAP must open DNA to permit transcription 3. RNAP must leave promoter—abortive initiation

  13. The Initiating Form of RNA Polymerase

  14.  (1) The discovery of initiation factors  factor is required for bacterial RNA polymerase to initiate transcription on promoters +  ' ' KD ~ 10-9 M } } ‘holoenzyme’ ‘core’ Can elongate but cannot begin transcription at promoters Can begin transcription on promoters and can elongate

  15. B. Initial purification Lysate various fractionation steps (DEAE column, glycerol gradient etc) Active fractions identified by assay How was discovered (Burgess, 1969) A. Assay for RNA polymerase: *ATP CTP GTP UTP E.coli lysate Calf thymus DNA buffer Look for incorporation of *ATP into RNA chains

  16. lysate Improved fractionation phosphocellulose column 2 Activity (*ATP) CT DNA 1 Peak 1 Peak 2 Fraction # '  increases rate of initiation  SDS gel analysis   Assay: incorporationPATP Transcription  DNA  g  C. Improved purification of RNA polymerase: Labmate Jeff Roberts reported that the new, improved preparation of RNAP (peak 2) had no activity on  DNA salt OD 280 Peak 1 restored activity

  17. (3) s undergoes a large conformational change upon binding to RNA polymerase Free  doesn’t bind DNA  in holoenzyme positioned for DNA recognition Sorenson; 2006

  18. Recognition of the prokaryotic promoter -35 logo -10 logo

  19. sis positioned for DNA recognition

  20. Initiating RNAP must open DNA to permit transcription: Formation of the open complex

  21. The Strand Separation/Melting Step -35 logo -10 logo Helix-turn-helix in Domain 4 Recognizes -35 as duplex DNA Is the -10 promoter element recognized as Duplex or SS DNA?

  22. Approach 1. Determine a high resolution structure ofs2 bound to non-template strand of the -10 element Schematic 2. Determine whether this structure represents the “initial binding state” or endpoint state

  23. Identifying eukaryotic “initiation factors”

  24. RNA Pol II + NTPs + DNA containing a real promoter NO TRANSCRIPTION promoter RNA Pol II + NTPs + DNA with real promoter nuclear extract TRANSCRIPTION INITIATION and ELONGATION Transcription Initiation by PolII requires many General Transcription Factors

  25. 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 1 Binds TATA box; higher eukaryotes have multiple TBPs TAFs ~10 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 2 Recruits TFIIH; Facilitates forming initiation-competent pol II; promoter clearance TFIIH 9 ATPase/kinase activity. Helicase: unwinds DNA at transcription startsite; kinase phosphorylates ser5 of RNA polymerase CTD; helps release RNAP from promoter Purification scheme for partially purified general transcription factors. Fractionation of HeLa 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)

  26. 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

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

  28. 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 (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.

  29. NTPs KB Kf Elongating Complex Abortive Initiation R+P RPc RPo initial binding “isomerization” Steps in transcription initiation

  30. NTPs KB Kf Elongating Complex Abortive Initiation R+P RPc RPo initial binding “isomerization” Abortive Initiation and Promoter escape During abortive initiation, RNAP synthesizes many short transcripts, but reinitiates rapidly. How can the active site of RNAP move forward along the DNA while maintaining contact with the promoter?

  31. Förster (fluorescence) resonance energy transfer (FRET) allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution Experimental set-up for single molecule FRET: Single transcription complexes labeled with a fluorescent donor (D, green) and a fluorescent acceptor (A, red) are illuminated as they diffuse through a femtoliter-scale observation volume (green oval; transit time ~1 ms); observed in confocal microscope Using single molecule FRET to monitor movement of RNAP and DNA

  32. Three models for Abortive initiation #1 Predicts movement of both the RNAP leading and trailing edge relative to DNA #2 Predicts expansion and contraction of RNAP #3 Predicts expansion and contraction of DNA

  33. Initial transcription involves DNA scrunching Open complex Lower E* peak is free DNA; higher E* peak is DNA in open complex; distance is shorter because RNAP induces DNA bending A. N. Kapanidis et al., Science 314, 1144 -1147 (2006)

  34. Initial transcription involves DNA scrunching Open complex Abortive initiation complex Higher E* in Abortive initiation complex than open complex results from DNA scrunching

  35. Initial transcription involves DNA scrunching Open complex Abortive initiation complex

  36. The energy accumulated in the DNA scrunched “stressed intermediate could disrupt interactions between RNAP,  and the promoter, thereby driving the transition from initiation to elongation At a typical promoter, promoter escape occurs only after synthesis of an RNA product ~9 to 11 nt in length (1–11) and thus can be inferred to require scrunching of ~7 to 9 bp (N – 2, where N = ~9 to 11; Fig. 3C). Assuming an energetic cost of base-pair breakage of ~2 kcal/mol per bp (30), it can be inferred that, at a typical promoter, a total of ~14 to 18 kcal/mol of base-pair–breakage energy is accumulated in the stressed intermediate. This free energy is high relative to the free energies for RNAP-promoter interaction [~7 to 9 kcal/mol for sequence-specific component of RNAP-promoter interaction (1)] and RNAP-initiation-factor interaction [~13 kcal/mol for transcription initiation factor {sigma}70 (31)].

  37. sis positioned to block elongating transcripts

  38. Validation of the prediction that  occlusion of the RNA exit channel promotes “abortive initiation” #1: transcription by holoenzyme with full-length  #2: transcription by holoenzyme with truncated at Region 3.2: lacks  in the RNA exit channel Murakami, Darst 2002

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