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Control of Transcription Initiation General References Chapter 16 of Molecular Biology of the Gene 6th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M, Losick, R. 547-587. Ptashne, M. and Gann, A. (2002) Genes and Signals. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Luscombe, N.M., Austin, S.E., Berman, H.M., Thornton, J.M. (2000) An overview of the structures of protein-DNA complexes. Genome Biology 1(1): reviews001.1-001.37 Examples of Control Mechanisms Alternative Sigma Factors Sorenson, MK, Ray, SS, Darst, SA (2004) Crystal structure of the flagellar sigma/anti-sigma complex 28 /FlgM reveals an intact sigma factor in an inactive conformation. Molecular Cell 14:127-138. Gruber, TM, Gross, CA (2003) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev. Microbiol 57:441-66 Increasing the Initial Binding of RNA Polymerase Holoenzyme to DNA Lawson CL, Swigon D, Murakami KS, Darst SA, Berman HM, Ebright RH. (2004) Catabolite activator protein: DNA binding and transcription activation. Curr Opin Struct Biol. 14:10-20. Increasing the Rate of Isomerization of RNA Polymerase *Dove, S.L., Huang, F.W., and Hochschild, A. (2000) Mechanism for a transcriptional activator that works at the isomerization step. Proc Natl Acad Sci USA 97: 13215-13220. Jain, D. Nickels, B.E., Sun, L., Hochschild, A., and Darst, S.A. (2004) Structure of a ternary transcription activation complex. Mol Cell 13: 45-53. Hawley and McClure (1982) Mechanism of Activation of Transcription from the l PRM promoter. JMB 157: 493-525 DNA looping **Oehler, S., Eismann, E.R., Kramer, H. and Mueller-Hill, B. (1990) The three operators of the lac operon cooperate in repression. EMBO 9:973979. Vilar, J.M.G. and Leibler, S. (2003) DNA looping and physical constraints on transcription regulation. J Mol Biol 331:981-989. Dodd, I.B., Shearwin, K.E., Perkins, A.J., Burr, T., Hochschild, A., and Egan, J.B. (2004) Cooperativity in long-range gene regulation by the l cI repressor. Genes Dev. 18:344-354. The dynamics of lac Repressor binding to its operator Elf, J., Li, G.W., and Xie, X.S. (2007). Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 1191–1194. Li, G.W., Berg, O.G., and Elf, J. (2009). Effects of macromolecular crowding and DNA looping on gene regulation kinetics. Nat. Phys. 5, 294–297 Li, G.W., and Xie, X.S. (2011). Central dogma at the single-molecule level in living cells. Nature 475, 308–315. Hammar, P., Leroy, P., Mahmutovic, A., Marklund, E.G., Berg, O.G., and Elf, J. (2012). The lac repressor displays facilitated diffusion in living cells. Science 336, 1595–1598 *Choi, PJ, Cai,L, Frieda K and X. Sunney Xie (2008) A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell Science 2008: 442-446. [DOI:10.1126/science.1161427] In vivo logic of absolute rates of protein synthesis Li, GW, Burkhardt D, Gross, C and Weissman JS (2014). Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell.157(3):624-35. doi: 10.1016 Proofreading *Zenkin, N, Yuzenkova, y Severinov K Transcript-assisted transcriptional proofreading. Science. 2006 Jul 28;313(5786):518-20 Sydow JF, Cramer P. (2009) RNA polymerase fidelity and transcriptional proofreading.Curr Opin Struct Biol. 2009 Dec;19(6):732-9. Epub 2009 Nov 13. Sydow JF, Brueckner F, Cheung AC, Damsma GE, Dengl S, Lehmann E, Vassylyev D, Cramer P.(2009) Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol Cell. Jun 26;34(6):710-21. Pausing Artsimovitch, I. and Landick, R (2000). Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals. PNAS 97: 7090-7095 Zhang J, Palangat M, Landick R. Role of the RNA polymerase trigger loop in catalysis and pausing. Nat Struct Mol Biol. 2010 Jan;17(1):99104. Epub 2009 Dec 6. *Shaevitz, j. Abbondanzieri E, Landick R. and Block S (2003) Backtracking by single RNA polymerase molecules observed at near base pair resolution. Nature 426: 684-687 Herbert, K., La Porta, A, Wong B, Mooney, R. Neuman, K. Landick, R. and Block, S.(2006). Sequence-Resolved Detection of Pausing by Single RNA Polymerase Molecules. Cell 125:1083-1094 *Weixlbaumer, A, Leon, K, Landick, R and Darst SA (2013) Structural basis of transcriptional pausing in bacteria. Cell. 2013 Jan 31;152(3):431-41. doi: 10.1016/j.cell.2012.12.020. Regulation through the 2˚ channel 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 Measurement of elongation Larson MH, Mooney RA, Peters JM, Windgassen T, Nayak D, Gross CA, Block SM, Greenleaf WJ, Landick R, Weissman JS. Science. 2014: A pause sequence enriched at translation start sites drives transcription dynamics in vivo. May 30;344(6187):1042-7. Shaevitz JW, Abbondanzieri EA, Landick R, Block SM molecules observed at near-base-pair resolution. Nature. 2003 Dec 11;426(6967):684-7. Epub 2003 Nov 23. Backtracking by single RNA polymerase Important Points 1. Every step in transcription initiation can be regulated to increase or decrease the number of successful initiations per time. 2. In E. coli, transcription initiation is controlled primarily by alternative factors and by a large variety of other sequence-specific DNA-binding proteins. 3. G=RTlnKD. This means that a net increase of 1.4 kcal/mole (the approximate contribution of an additional hydrogen bond) increases binding affinity by 10-fold. Many examples of transcription activation in bacteria take advantage of such weak interactions. 4. To activate transcription at a given promoter by increasing KB, the concentration of RNA polymerase in the cell and its affinity for the promoter must be in the range so an increase in KB makes a difference. Likewise, to activate transcription by increasing kf, the rate of isomerization must be slow enough so the increase makes a substantial difference. 5. Network motifs give the regulatory circuit its properties 6. Transcriptional pauses are integral to the transcription process and are extensively utilized for regulatory roles Transcriptional Control: Bacterial Paradigms Every step of transcription can be regulated NTPs KB R+P RPc initial binding Kf RPo Abortive Initiation “isomerization” DNA Binding Proteins used to alter promoter properties Elongating Complex How proteins recognize DNA All 4 bp can be distinguished in the major groove Common families of DNA binding proteins Gene regulation in E. coli: The Broad Perspective • 4400 genes • 300-350 sequence-specific DNA-binding proteins • 7 factors In E. coli 1 copy/cell ≈ 10-9 M If KD = 10-9M and things are simple: 10 copies/cell 100 copies/cell occupied 90% occupied 99% Regulation by repressors and activators Case Study: How bacteria monitor and respond to nutrient status Regulation of the lactose utilization operon: Dual negative and positive control A P O lacY lacZ lacA Repressor Activator CAP-cAMP NTPs KB R+P RPc initial binding Kf RPo “isomerization” Abortive Initiation Lac repressor and DNA looping Lac ~ 1980 -35 -10 Lac operator Lac 2000 O3 -90 O1 -35 -10 O2 +400 Oehler, 2000 O2 1/10 affinity of O1 O3 1/300 affinity of O1 What is the function of these weak operators? The weak operators significantly enhance represssion Oehler, 2000 Through DNA looping, Lac repressor binding to a “strong” operator (Om) can be helped by binding to a “weak” operator (OA) OK Om Oa Better! Om A mutant Lac repressor that cannot form tetramers is not helped by a weak site MM Effects of looping (2 operators) Om (main operator) binds repressor more tightly than Oa (auxiliary operator). Transcription takes place only in the states (i) and (iii), when Om is not occupied. Vilar, J.M.G. and Leibler, S. (2003) J Mol Biol 331:981-989 One operator: a single unbinding event is enough for the repressor to completely leave the neighborhood of the main operator. Two operators: repressor can escape the neighborhood of the main operator only if it sequentially unbinds both operators. Allows control of gene regulation on multiple time scales through different kinds of dissociation events Partial dissociation: can initiate 1round of transcription (~10-20 molecules) Full dissociation: 6 min to find site again; allows establishing bistability I. Activating transcription initiation at KB (initial binding) step Positive control: activators ( e.g. CAP); facilitate RNAP binding with favorable protein-protein contact Favorable contact A * RNAP holo -35 -10 ∆ G = RT lnKD; if * nets 1.4 kcal/mol, KB goes up 10-fold Activating by increasing KB is effective only if initial promoter occupancy is low If favorable contact nets 1.4Kcal/mole (KB goes up 10X) then: a) If initial occupancy of promoter is low RNAP A * RNAP 10% occupied 1% occupied Transcription rate increases 10-fold b) If initial occupancy of promoter is high RNAP 99% occupied A * RNAP 99.9% occupied Little or no effect on transcription rate A case study of activation at KB: CAP at the lac operon: How is CAP activated? cAMP Inactive CAP high glucose Active CAP Regulates >100 genes positively or negatively CAP at lac operon CAP increases transcription ~40-fold; KB ; no effect on kf Strategies to identify point of contact between CAP and RNAP 1. Isolate “positive control” (pc) mutations in CAP. These mutant proteins bind DNA normally but do not activate transcription M M 2. “Label transfer” (in vitro) from activator labeled near putative “pc” site to RNAP Activate X*; reduce S-S; X* is transferred to nearest site; determine location by protein cleavage studies; X* transferred to -CTD 3. Isolate CAP-non-responsive mutations in -CTD S-S-X* RNAP -35 -10 M RNAP -35 -10 Summary: Stereotypical binding of repressors and activators regulates transcription initiation Negative control: repressors (e.g. l, Lac ); prevent RNAP binding R -35 -10 Positive control: activators ( e.g. CAP); facilitate RNAP binding with favorable protein-protein contact A Favorable contact * RNAP holo -35 NTPs KB R+P RPc initial binding -10 Kf RPo “isomerization” Abortive Initiation Elongating Complex Regulatory Circuits are composed of network motifs Negative feedback loops: tunes expression to cellular state Blue line: negative feedback Red line: constant rate of A synthesis unaffected by R Positive feed back loops can generate bistability Combinatorial control of gene expression AND NOT Logic, e.g. lac operon AND Logic; e.g. arabinose operon Regulated Elongation Transcriptional pauses are really important Coordinate transcription (RNAP movement) with: 1) Folding nascent RNA 2) Other RNA processes translation, degradation, export, splicing 3) Regulator binding (TAR—HIV; RfaH prokaryotes) Promoter proximal pauses poise RNAPII for gene expression in metazoans How to measure pauses Time (Min) Run-off transcript-- Pause transcript-- Stall (3 NTP’s) Pauses are characterized by duration and “efficiency” (probability of entering the pause state at kinetic branch between pausing and active elongation) Start reaction with 4th *NTP + heparin to prevent reinitiation Aliquots of a synchronized, radiolabeled, single-round transcription assay were removed at various times and electrophoresed on a polyacrylamide gel; separation by size Pauses can also be measured using single molecule technology Pausing can also be measured using single molecule techniques Can follow single molecules over long times and detect very short pauses Identification of Elemental pauses Trace of two RNA polymerase molecules, one with long pause Backtracking by eye: phase 1 (backtracking, solid line) phase 2 (pause, dotted line) phase 3 (recovery, solid line). Representative short pause (3 s); No backtracking *Short pauses account for 95% of all pausing events; subsequent studies confirmed that they are not backtracked and occur at specific sequences (ubiquitous/elemental pauses) Pauses can also be measured genome wide using NET-seq Matt Larson ( Weissman lab) Current view of Pausing (?) Elemental Pause Elongation Complex Regulating Termination: Attenuation control 1. Stabilizing alternative 2˚structures of mRNA can lead to either elongation or termination 2. External inputs can alter the equilibrium between mRNA states 3. RNA polymerase pausing is critical for this regulatory mechanism Attenuation in biosynthetic operons His codons TAA hisL hisG 1 2 No protein synthesis hisL 3 1 2 4 hisG 3 4 pause transcription hairpin terminator High His TAA hisL 3 4 hisG 2 transcription terminator 1 Operon mRNA level TAA Low His hisL 2 3 1 transcription4 anti-terminator Low hisG High Regulated “attenuation” (termination) is widespread Switch between the “antitermination” and “termination” Stem-loop structures can be mediated by: 1. Ribosome pausing ( reflects level of a particular charged tRNA): regulates expression of amino acid biosynthetic operons in gram - bacteria 2. Uncharged tRNA: promotes anti-termination stem-loop in amino acyl tRNA synthetase genes in gm + bacteria 3. Proteins: stabilize either antitermination or termination stem-loop structures 4. Small molecules: aka riboswitches 5. Alternative 2˚ structures can also alter translation, self splicing, degradation E. coli NusG: A 21kD essential elongation factor NTD NGN domain CTD KOW domain Activities: 1. Increases elongation rate 2. suppresses backtracking 3. Required for anti-termination mechanisms 4. Enhances termination mediated by the rho-factor How does one 21Kd protein mediate all of these activities? The CTD of NusG interacts with other protein partners NusG CTD 50 µM NusE Rho NusE, a ribosomal protein (S10) is part of a complex of proteins mediating antitermination/termination depending on its protein partners Rho is an RNA binding hexamer that mediates termination by dissociating RNA from its complex with RNA polymerase and DNA using stepwise physical forces on the RNA derived from alternating protein conformations coupled to ATP hydrolysis Although the CTD mediates the protein interactions involved in termination and antitermination, full length NusG is required for both processes, presumably because NusG must be tethered to RNA polymerase for these functions Coupled syntheses. J W Roberts Science 2010;328:436-437 Published by AAAS NusG, the only universal elongation factor, exhibits divergent interactions with other regulators