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Biochemistry 201 Biological Regulatory Mechanisms Transcription and Its Regulation

Biochemistry 201 Biological Regulatory Mechanisms Transcription and Its Regulation January 22 –Mechanism of Transcription Initiation January 24– Mechanism of Transcription Elongation January 28– Control of Transcription in Bacteria January 31– Control of Transcription in Eukaryotes

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Biochemistry 201 Biological Regulatory Mechanisms Transcription and Its Regulation

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  1. Biochemistry 201 Biological Regulatory Mechanisms Transcription and Its Regulation January 22 –Mechanism of Transcription Initiation January 24– Mechanism of Transcription Elongation January 28– Control of Transcription in Bacteria January 31– Control of Transcription in Eukaryotes 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. 2. Reviews Murakami KS, Darst SA. (2003) Bacterial RNA polymerases: the wholo story. Curr Opin Struct Biol 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 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 Natl Acad Sci 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 ***Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, Foster JW, Gourse RL. (2004) DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. Cell. 6:311-22. The accompanying minireview is helpful Nickels, B.E. and Hochschild, A. (2004) Regulation of RNA Polymerase through the Secondary Channel. Cell118:281-284

  3. 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

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

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

  6. I. RNA polymerases

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

  8. 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

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

  10. 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

  11. The Initiating Form of RNA Polymerase

  12.  (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

  13. 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

  14. 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

  15. There are several flavors of promoters  and  recruit RNAP to promoter DNA (2) Bacterial promoters

  16. (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

  17. s is positioned for DNA recognition

  18. s is positioned to affect key activities of RNA polymerase

  19. Surprising structural similarity between the initiating forms of bacterial and eukaryotic RNAP

  20. 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.

  21. TFIIB structure TFIIB has a central role in initiation similar to that of  Recruits Pol II to promoter: N-terminus binds Pol II; C terminus binds TBP and DNA Role in promoter opening; B linker mutants recruit PolII but cant strand open or initiate Role in selection of TSS ( Inr): B reader mutants Blocks elongating RNAchain: B reader Crystal structure of TFB + RNA polymerase--archae D Kostrewa et al.Nature462, 323-330 (2009) doi:10.1038/nature08548

  22. Topological similarities in /TFIIB binding to RNAP B ribbon (4):both bind flap tip helix B linker (2): both bind coiled -coil and rudder; both involved in strand opening B core (3) B reader ( 3.2): both in exit channel and near active site; start site selection D Kostrewa et al.Nature462, 323-330 (2009) doi:10.1038/nature08548 TFIIB and  bound to RNA polymerase show surprising similarity. Analogously placed regions have similar functions

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

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

  25. A detailed look at a prokaryotic promoter 15-19nucleotides T T G A C A T A T A A T -35 -10 Sequence Logos -35 logo -10 logo

  26. Recognition of the prokaryotic promoter -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?

  27. Approach 1. Determine a high resolution structure of s2 bound to non-template strand of the -10 element Schematic 2. Determine whether this structure represents the “initial binding state” or endpoint state

  28. Promoter escape

  29. s is positioned to affect key activities of RNA polymerase

  30. Promoter escape and Abortive Initiation 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 promoter contact?

  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. 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

  38. s is positioned to affect key activities of RNA polymerase

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