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Site-Specific Incorporation of Probes into RNA Polymerase by Unnatural-Amino-Acid Mutagenesis and Staudinger–Bertozzi Ligation

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Bacterial Transcriptional Control

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1276))

Abstract

A three-step procedure comprising (1) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (2) Staudinger–Bertozzi ligation with a probe-phosphine derivative, and (3) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a cross-linking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.

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References

  1. Miyake R, Murakami K, Owens J, Greiner D, Ozoline O, Ishihama A, Meares C (1998) Dimeric association of Escherichia coli RNA polymerase α subunits, studied by cleavage of single-cysteine alpha subunits conjugated to iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate. Biochemistry 37:1344–1349

    Article  CAS  PubMed  Google Scholar 

  2. Owens J, Chmura A, Murakami K, Fujita N, Ishihama A (1998) Mapping the promoter DNA sites proximal to conserved regions of σ70 in an Escherichia coli RNA polymerase-lacUV5 open promoter complex. Biochemistry 37:7670–7675

    Article  CAS  PubMed  Google Scholar 

  3. Chen Y, Ebright Y, Ebright RH (1994) Identification of the target of a transcription activator protein by protein-protein photocrosslinking. Science 265:90–92

    Article  CAS  PubMed  Google Scholar 

  4. Miller A, Wood D, Ebright RH, Rothman-Denes L (2004) RNA polymerase beta′ subunit: a target of DNA binding-independent activation. Science 75:1655–1657

    Google Scholar 

  5. Callaci S, Heyduk E, Heyduk T (1998) Conformational changes of Escherichia coli RNA polymerase σ70 factor induced by binding to the core enzyme. J Biol Chem 273:32995–33001

    Article  CAS  PubMed  Google Scholar 

  6. Callaci S, Heyduk E, Heyduk T (1999) Core RNA polymerase from E. coli induces a major change in the domain arrangement of the σ70 subunit. Mol Cell 3:229–238

    Article  CAS  PubMed  Google Scholar 

  7. Heyduk E, Heyduk T (1999) Architecture of a complex between the σ70 subunit of Escherichia coli RNA polymerase and the nontemplate strand oligonucleotide. J Biol Chem 274:3315–3322

    Article  CAS  PubMed  Google Scholar 

  8. Mukhopadhyay J, Kapanidis A, Mekler V, Kortkhonjia E, Ebright YW, Ebright RH (2001) Translocation of σ70 with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA. Cell 106:453–463

    Article  CAS  PubMed  Google Scholar 

  9. Mekler V, Kortkhonjia E, Mukhopadhyay J, Knight J, Revyakin A, Kapanidis A, Niu W, Ebright YW, Levy R, Ebright RH (2002) Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cell 108:599–614

    Article  CAS  PubMed  Google Scholar 

  10. Mukhopadhyay J, Mekler V, Kortkhonjia E, Kapanidis A, Ebright YW, Ebright RH (2003) Fluorescence resonance energy transfer (FRET) in analysis of transcription-complex structure and function. Methods Enzymol 371:144–159

    Article  CAS  PubMed  Google Scholar 

  11. Mukhopadhyay J, Sineva E, Knight J, Levy R, Ebright RH (2004) Antibacterial peptide microcin J25 inhibits transcription by binding within, and obstructing, the RNA polymerase secondary channel. Mol Cell 14:739–751

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Knight J, Mekler V, Mukhopadhyay J, Ebright RH, Levy R (2005) Distance-restrained docking of rifampicin and rifamycin SV to RNA polymerase using systematic FRET measurements: developing benchmarks of model quality and reliability. Biophys J 88:925–938

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Kapanidis A, Margeat E, Laurence T, Doose S, Ho S, Mukhopadhyay J, Kortkhonjia E, Mekler V, Ebright RH, Weiss S (2005) Retention of transcription Initiation factor σ70 in transcription elongation: single-molecule analysis. Mol Cell 20:347–356

    Article  CAS  PubMed  Google Scholar 

  14. Margeat E, Kapanidis A, Tinnefeld P, Wang Y, Mukhopadhyay J, Ebright RH, Weiss S (2006) Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes. Biophys J 90:1419–1431

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Kapanidis A, Margeat E, Ho S, Kortkhonjia E, Weiss S, Ebright RH (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314:1144–1147

    Article  PubMed Central  PubMed  Google Scholar 

  16. Chakraborty A, Wang D, Ebright YW, Korlann Y, Kortkhonjia E, Kim T, Chowdhury S, Wigneshweraraj S, Irschik H, Jansen R, Nixon BT, Knight J, Weiss S, Ebright RH (2012) Opening and closing of the bacterial RNA polymerase clamp. Science 337:591–595

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Ebright Y, Chen Y, Pendergrast PS, Ebright R (1992) Incorporation of an EDTA-metal complex at a rationally selected site within a protein: application to EDTA-iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro. Biochemistry 31:10664–10670

    Article  CAS  PubMed  Google Scholar 

  18. Igarashi K, Ishihama A (1991) Bipartite functional map of the E. coli RNA polymerase α subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell 65:1015–1022

    Article  CAS  PubMed  Google Scholar 

  19. Kashlev M, Martin E, Polyakov A, Severinov K, Nikiforov V, Goldfarb A (1993) Histidine-tagged RNA polymerase: dissection of the transcription cycle using immobilized enzyme. Gene 130:9–14

    Article  CAS  PubMed  Google Scholar 

  20. Tang H, Severinov K, Goldfarb A, Ebright RH (1995) Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A 92:4902–4906

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Tang H, Kim Y, Severinov K, Goldfarb A, Ebright RH (1996) Escherichia coli RNA polymerase holoenzyme: rapid reconstitution from recombinant α, β, β′, and σ subunits. Methods Enzymol 273:130–134

    Article  CAS  PubMed  Google Scholar 

  22. Naryshkin N, Kim Y, Dong Q, Ebright RH (2001) Site-specific protein-DNA photocrosslinking: analysis of bacterial transcription initiation complexes. Methods Mol Biol 148:337–361

    CAS  PubMed  Google Scholar 

  23. Naryshkin N, Druzhinin S, Revyakin A, Kim Y, Mekler V, Ebright RH (2009) Static and kinetic site-specific protein-DNA photocrosslinking: analysis of bacterial transcription initiation complexes. Methods Mol Biol 543:403–437

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Severinov K, Muir T (1998) Expressed protein ligation, a novel method for studying protein-protein interactions in transcription. J Biol Chem 273:16205–16209

    Article  CAS  PubMed  Google Scholar 

  25. Severinov K, Mustaev A, Severinova E, Bass I, Kashlev M, Landick R, Nikiforov V, Goldfarb A, Darst S (1995) Assembly of functional Escherichia coli RNA polymerase containing β subunit fragments. Proc Natl Acad Sci U S A 92:4591–4595

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Severinov K, Mustaev A, Kukarin A, Muzzin O, Bass I, Darst S, Goldfarb A (1996) Structural modules of the large subunits of RNA polymerase. Introducing archaebacterial and chloroplast split sites in the β and β′ subunits of Escherichia coli RNA polymerase. J Biol Chem 271:27969–27974

    Article  CAS  PubMed  Google Scholar 

  27. Grohmann D, Nagy J, Chakraborty A, Klose D, Fielden D, Ebright RH, Michaelis J, Werner F (2011) The initiation factor TFE and the elongation factor Spt4/5 compete for the RNAP clamp during transcription initiation and elongation. Mol Cell 43:263–274

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Chin J, Santoro S, Martin A, King D, Wang L, Schultz P (2002) Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc 124:9026–9027

    Article  CAS  PubMed  Google Scholar 

  29. Young T, Ahmad I, Yin J, Schultz P (2009) An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol 395:361–374

    Article  PubMed  Google Scholar 

  30. Saxon E, Bertozzi C (2000) Cell surface engineering by a modified Staudinger reaction. Science 287:2007–2010

    Article  CAS  PubMed  Google Scholar 

  31. Kiick K, Saxon E, Tirrell D, Bertozzi C (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc Natl Acad Sci U S A 99:19–24

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Chakraborty A, Wang D, Ebright Y, Ebright RH (2010) Azide-specific labeling of biomolecules by Staudinger-Bertozzi ligation: phosphine derivatives of fluorescent probes suitable for single-molecule fluorescence spectroscopy. Methods Enzymol 472:19–30

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Peter Schultz and Ryan Mehl for plasmids. This work was supported by National Institutes of Health grant GM041376 and a Howard Hughes Medical Institute Investigatorship to R.H.E.

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Authors and Affiliations

  1. Waksman Institute and Department of Chemistry and Chemical Biology, Rutgers University, 190 Frelinghuysen Road, Piscataway, NJ, 08854, USA

    Anirban Chakraborty, Abhishek Mazumder, Miaoxin Lin, Adam Hasemeyer, Qumiao Xu, Dongye Wang, Yon W. Ebright & Richard H. Ebright

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  1. Anirban Chakraborty
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  2. Abhishek Mazumder
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  3. Miaoxin Lin
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  4. Adam Hasemeyer
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  5. Qumiao Xu
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  6. Dongye Wang
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  7. Yon W. Ebright
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  8. Richard H. Ebright
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Corresponding author

Correspondence to Richard H. Ebright .

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Editors and Affiliations

  1. Department of Microbiology, The Ohio State University, Columbus, Ohio, USA

    Irina Artsimovitch

  2. Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, USA

    Thomas J. Santangelo

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Chakraborty, A. et al. (2015). Site-Specific Incorporation of Probes into RNA Polymerase by Unnatural-Amino-Acid Mutagenesis and Staudinger–Bertozzi Ligation. In: Artsimovitch, I., Santangelo, T. (eds) Bacterial Transcriptional Control. Methods in Molecular Biology, vol 1276. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2392-2_6

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  • DOI: https://doi.org/10.1007/978-1-4939-2392-2_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2391-5

  • Online ISBN: 978-1-4939-2392-2

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