Properties of Escherichia coli RNA polymerase from a strain devoid of the stringent response alarmone ppGpp

• Authors: Agnieszka Szalewska-Pałasz
• Languages of publication: EN

Abstracts

EN

The stringent response alarmone guanosine tetraphosphate (ppGpp) affects transcription from many promoters. ppGpp binds directly to the transcription enzyme of Escherichia coli, RNA polymerase. Analysis of the crystal structure of RNA polymerase with ppGpp suggested that binding of this nucleotide may result in some conformational or post-translational alterations to the enzyme. These changes might affect in vitro performance of the enzyme. Here, a comparison of the in vitro properties of RNA polymerases isolated from wild type and ppGpp-deficient bacteria shows that both enzymes do not differ in i) transcription activity of various promoters (e.g. σ70-rrnB P1, λpL, T7A1), ii) response to ppGpp, iii) promoter-RNA polymerase open complex stability. Thus, it may be concluded that ppGpp present in the bacterial cell prior to purification of the RNA polymerase does not result in the alterations to the enzyme that could be permanent and affect its in vitro transcription capacity.

Keywords

EN

RNA polymerase; ppGpp; stringent response; transcription

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2008
• Volume: 55
• Issue: 2
• Pages: 317-323

Dates

published

2008

received

2008-03-25

revised

2008-04-29

accepted

2008-06-10

(unknown)

2008-06-14

Contributors

author

Agnieszka Szalewska-Pałasz

• Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland

References

• Artsimovitch I, Patlan V, Sekine S, Vassylyeva MN, Hosaka T, Ochi K, Yokoyama S, Vassylyev DG (2004) Structural basis for transcription regulation by alarmone ppGpp. Cell 117: 299-310.
• Barker MM, Gaal T, Josaitis CA, Gourse RL (2001) Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro. J Mol Biol 305: 673-688.
• Bartlett MS, Gaal T, Ross W, Gourse RL (1998) RNA polymerase mutants that destabilize RNA polymerase-promoter complexes alter NTP-sensing by rrn P1 promoters. J Mol Biol 279: 331-345.
• Bernardo LMD, Johansson L, Solera D, Skarfstad E, Shingler V (2006) The ppGpp alarmone, DksA, and promoter affinity for RNA polymerase in regulation of σ^54-dependent transcription. Mol Microbiol 60: 749-764.
• Burgess RR, Jendrisak JJ (1975) A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry 14: 4634-4638.
• Burgess RR, Erickson B, Gentry D, Gribskov M, Hager D, Lesley S, Strickland M, Thompson N (1987) Bacterial RNA polymerase subunits and genes. In: RNA Polymerase and the Regulation of Transcription. Reznikoff WS, Burgess RR, Dahlberg JE, Gross CA, Record MT Jr, Wickens MP, eds, pp 3-15. Elsevier Science Publications Co., Inc.
• Cashel M (1974) Preparation of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) from Escherichia coli ribosomes. Anal Biochem 57: 100-107.
• Cashel M, Gentry D, Hernandez VJ, Vinella D (1996) The stringent response. In Escherichia coli and Salmonella: Cellular and Molecular Biology, vol 1, pp 1458-1496. American Society for Microbiology, Washington DC.
• Chatterji D, Fujita N, Ishihama A (1998) The mediator for stringent control, ppGpp, binds to the beta-subunit of Escherichia coli RNA polymerase. Genes Cells 3: 279-287.
• Fujita N, Ishihama A (1996) Reconstitution of RNA polymerase. Methods Enzymol 273: 121-130.
• Gaal T, Ross W, Estrem ST, Nguyen LH, Burgess RR, Gourse RL (2001) Promoter recognition and discrimination by EσS RNA polymerase. Mol Microbiol 42: 939-954.
• Gentry DR, Cashel M (1995) Mutational analysis of the Escherichia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation. Mol Microbiol 19: 1373-1384.
• Goff CG (1984) Coliphage-induced ADP-ribosylation of Escherichia coli RNA polymerase. Methods Enzymol 106: 418-429.
• Hager DA, Jin DJ, Burgess RR (1990) Use of Mono Q high-resolution ion-exchange chromatography to obtain highly pure and active Escherichia coli RNA polymerase. Biochemistry 29: 7890-7894.
• Hernandez VJ, Bremer H (1991) Escherichia coli ppGpp synthetase II activity requires spoT. J Biol Chem 266: 5991-5999.
• Jasiecki J, Węgrzyn G (2006) Phosphorylation of Escherichia coli poly(A) polymerase I and effects of this modification on the enzyme activity. FEMS Microbiol Lett 261: 118-122.
• Jishage M, Kvint K, Shingler V, Nyström T (2002) Regulation of σ-factor competition by the alarmone ppGpp. Genes Dev 16: 1260-1270.
• Kusano S, Ishihama A (1997) Functional interaction of Escherichia coli RNA polymerase with inorganic polyphosphate. Genes Cells 2: 433-441.
• Kvint K, Farewell A, Nystrom T (2000) RpoS-dependent promoters require guanosine tetraphosphate for induction even in the presence of high level of σ^S. J Biol Chem 275: 14795-14798.
• Laurie A, Bernardo LM, Sze CC, Skarfstad E, Szalewska-Pałasz A, Nystrom T, Shingler V (2003) The role of the alarmone (p)ppGpp in σ^N competition for core RNA polymerase. J Biol Chem 278: 1494-1503.
• Najmanová L, Janata J, Kopecký J, Spízek J (2003) Spore-specific modification of DNA-dependent RNA polymerase alpha subunit in streptomycetes - a new model of transcription regulation. Folia Microbiol (Praha) 48: 573-579.
• O'Neill E, Wikstrom P, Shingler V (2001) An active role for a structured B-linker in effector control of the σ^54-dependent regulator DmpR. EMBO J 20: 819-827.
• Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, Foster JW, Gourse RL (2004a) DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. Cell 118: 311-322.
• Paul BJ, Ross W, Gaal T, Gourse RL (2004b) rRNA transcription in Escherichia coli. Annu Rev Genet 38: 749-770.
• Paul BJ, Berkmen MB, Gourse RL (2005) DksA potentiates direct activation of amino acid promoters by ppGpp. Proc Natl Acad Sci USA 102: 7823-7828.
• Perederina A, Svetlov V, Vassylyeva MN, Tahirov TH, Yokoyama S, Artsimovitch I, Vassylyev DG (2004) Regulation through the secondary channel. Structural framework for ppGpp-DksA synergism during transcription. Cell 118: 297-309.
• Reynolds R, Bermudez-Cruz RM, Chamberlin MJ (1992) Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators. J Mol Biol 224: 31-51.
• Rohrer H, Zillig W, Mailhammer R (1975) ADP-ribosylation of DNA-dependent RNA polymerase of Escherichia coli by an NAD^+: protein ADP-ribosyltransferase from bacteriophage T4. Eur J Biochem 60: 227-238.
• Schneider DA, Gaal T, Gourse RL (2002) NTP-sensing by rRNA promoters in Escherichia coli is direct. Proc Natl Acad Sci USA 99: 8602-8607.
• Sen R, King RA, Weisberg RA (2001) Modification of the properties of elongating RNA polymerase by persistent association with nascent antiterminator RNA. Mol Cell 7: 993-1001.
• Shingler V (2003) Integrated regulation in response to aromatic compounds: from signal sensing to attractive behaviour. Environ Microbiol 5: 1226-1241.
• Szalewska-Pałasz A, Węgrzyn G, Węgrzyn A (2007a) Mechanisms of physiological regulation of RNA synthesis in bacteria: new discoveries breaking old schemes. J Appl Genet 48: 281-294.
• Szalewska-Palasz A, Johansson LUM, Bernardo LMD, Skärfstad E, Stec E, Brännström K, Shingler V (2007b) Properties of RNA polymerase bypass mutants: implications for ppGpp- and DksA-mediated control of σ^54-dependent transcription. J Biol Chem 282: 18046-18056.
• Sze CC, Shingler V (1999) The alarmone (p)ppGpp mediates physiological-responsive control at the σ^54-dependent pO promoter. Mol Microbiol 31: 1217-1228.
• Sze CC, Laurie AD, Shingler V (2001) In vivo and in vitro effects of integration host factor at the DmpR-regulated sigma(54)-dependent pO promoter. J Bacteriol 183: 2842-2851.
• Toulokhonov II, Shulgina I, Hernandez VJ (2001) Binding of the transcription effector ppGpp to Escherichia coli RNA polymerase is allosteric, modular, and occurs near the N terminus of the β'-subunit. J Biol Chem 276: 1220-1225.
• Vrentas CE, Gaal T, Berkmen MB, Rutherford ST, Haugen SP, Ross W, Gourse RL (2008) Still looking for the magic spot: the crystallographically defined binding site for ppGpp on RNA polymerase is unlikely to be responsible for rRNA transcription regulation. J Mol Biol 377: 551-564.
• Xiao H, Kalman M, Ikehara K, Zemel S, Glaser G, Cashel M (1991) Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutant can be eliminated by spoT null mutations. J Biol Chem 266: 5980-5990.
• Zhang G, Campbell EA, Minakhin L, Richter C, Severinov K, Darst SA (1999) Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 A resolution. Cell 98: 811-824.