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RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs

利用非典型启动核苷酸（NCIN）进行转录起始的RNA加帽：利用NCIN和NTP测定转录起始的相对效率

JB Jeremy G. Bird
BN Bryce E. Nickels
Richard H. Ebright Richard H. Ebright

发布: 2017年06月20日第7卷第12期 DOI: 10.21769/BioProtoc.2336 浏览次数: 9499

评审: Gal HaimovichMelike ÇağlayanAnonymous reviewer(s)

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Abstract

It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and 3’-desphospho-coenzyme A (dpCoA), can serve as ‘non-canonical initiating nucleotides’ (NCINs) for transcription initiation by bacterial and eukaryotic cellular RNA polymerases (RNAPs) and that the efficiency of the reaction is determined by promoter sequence (Bird et al., 2016). Here we describe a protocol to quantify the relative efficiencies of transcription initiation using an NCIN vs. transcription initiation using a nucleoside triphosphate (NTP) for a given promoter sequence.

Keywords: RNA polymerase (RNA聚合酶) search Transcription (转录) search Non-canonical initiating nucleotide (NCIN) (非典型起始核苷酸（NCIN）) search RNA capping (RNA加帽) search ab initio RNA capping (从头开始RNA加帽) search NAD+ (NAD+) search NADH (NADH) search 3’-desphospho coenzyme A (3'-去磷酸辅酶A) search

Background

Transcription in bacteria, archaea, and eukaryotes is carried out by multi-subunit RNA polymerases (RNAPs) conserved in sequence, structure, and mechanism (Ebright, 2000; Lane and Darst, 2010). To initiate transcription, RNAP, together with one or more initiation factors, binds to a specific DNA sequence referred to as a ‘promoter’ and unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing an unwound ‘transcription bubble’ (Figure 1A; Ruff et al., 2015). RNAP then selects a transcription start site by expanding (‘scrunching’) or contracting (‘antiscrunching’) the transcription bubble to place transcription-start-site nucleotides in the RNAP active-center initiating site (‘i site’) and extending site (‘i+1 site’), binds a complementary initiating nucleotide substrate in the i site and a complementary extending substrate in the ‘i+1’ site, and catalyzes phosphodiester-bond formation to yield an initial RNA product (Winkelman et al., 2016).

In standard de novo transcription initiation, the initiating substrate is a nucleoside triphosphate (NTP), typically ATP or GTP (Nickels and Dove, 2011). However, recently it has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and 3’-desphospho-coenzyme A (dpCoA), can serve as alternative initiating substrates (‘non-canonical initiating nucleotides’; NCINs), yielding NCIN-capped RNA products that have distinctive 5’-end structures, stabilities, and translation efficiencies (Figures 1B-1C; Bird et al., 2016; Barvik et al., 2016; Jiao et al., 2017; Walters et al., 2017). It further has been established that the relative efficiencies of NCIN-mediated initiation vs. NTP-mediated initiation are determined by promoter sequence (Bird et al., 2016).

Here, we describe a protocol to determine the relative efficiencies of NCIN-mediated transcription initiation versus ATP-mediated transcription initiation, (kcat/KM, NCIN)/(kcat/KM, ATP), for a given promoter sequence. The protocol involves generating radiolabeled initial RNA products in a set of transcription reactions having a constant concentration of NCIN and varying concentrations of ATP, followed by quantifying NCIN-initiated RNA and total RNA, followed by plotting observed ratios of NCIN-initiated RNA to total RNA as a function of ratios of NCIN concentration to ATP concentration.


Figure 1. Transcription initiation. A. RNAP-promoter open complex (RPo) with unwound transcription bubble. Gray, RNAP; blue, -10-element nucleotides; i and i+1, RNAP active-center initiating nucleotide binding site and extending nucleotide binding site; boxes, DNA nucleotides (nontemplate-strand nucleotides above template-strand nucleotides). B. Structures of ATP and NAD+, Red, identical atoms in ATP and NAD+; C. Initial RNA products formed in transcription initiation using ATP (top) or transcription initiation using NAD+ (bottom). Left subpanels show initiating ATP or NAD+ bound in i site; right subpanels show initial RNA products formed using CTP as extending nucleotide. Red boxes, adenosine and cytosine moieties of ATP, NAD+, and CTP; green boxes, nicotinamide-riboside moiety of NAD+.

Materials and Reagents

  1. E. coli RNA polymerase σ70 holoenzyme
    Note: Prepared as in Mukhopadhyay et al. (2003) or purchased (New England Biolabs, catalog number: M0551S )
  2. E. coli RNA polymerase core enzyme
    Note: Prepared as in Artsimovitch et al. (2003).
  3. E. coli σ70
    Note: Prepared as Marr and Roberts (1997); Perdue and Roberts (2010).
  4. NAD+ (grade I, free acid) (Roche Molecular Systems, catalog number: 10127965001 )
  5. NADH (grade I, free acid) (Roche Molecular Systems, catalog number: 10107735001 )
  6. Phusion Flash HF master mix (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F548L )
    Note: For generating transcription templates.
  7. Oligodeoxyribonucleotides (template and primers) (Integrated DNA Technologies, www.IDTdna.com)
  8. QIAquick PCR purification kit (QIAGEN, catalog number: 28106 )
  9. TEMED (Avantor Performance Materials, J.T. Baker®, catalog number: 4098-01 )
  10. Ammonium persulfate (VWR, AMRESCO, catalog number: 97064-594 )
  11. GeneMate LE Quick-Dissolve agarose (BioExpress, catalog number: E-3119-500 )
  12. 3’-Desphosphocoenzyme A (Sigma-Aldrich, catalog number: D3385 )
  13. SequaGel sequencing system (National Diagnostics, catalog number: EC-833 )
  14. High purity rNTP set (ATP, UTP, GTP, CTP) (100 mM) (GE Healthcare, catalog number: 27-2025-01 )
  15. [α-32P]-CTP EasyTide (3,000 Ci/mmol) (250 μCi) (Perkin Elmer, catalog number: BLU508H250UC )
  16. Tris base (VWR, AMRESCO, catalog number: 97061-800 )
  17. Potassium chloride (KCl) (EMD Millipore, catalog number: PX1405-1 )
  18. Magnesium chloride hexahydrate (EMD Millipore, catalog number: 5980-500GM )
  19. EDTA disodium salt dyhydrate (1 kg) (VWR, AMRESCO, catalog number: 97061-018 )
  20. Dithiothreitol (DTT) (Gold Bio, catalog number: DTT50 )
  21. Bovine serum albumin (BSA) fraction V (Alfa Aesar, Affymetrix/USB, catalog number: J10857 )
  22. Sodium dodecylsulfate (SDS) (VWR, AMRESCO, catalog number: 97064-470 )
  23. Deionized formamide (EMD Millipore, catalog number: 4610-100ML )
  24. Xylene cyanol (Sigma-Aldrich, catalog number: X4126-10G )
  25. Bromophenol blue (EMD Millipore, catalog number: BX1410-7 )
  26. Amaranth red (Acros Organics, catalog number: AC15303-0250 )
  27. Boric acid (ACS grade) (VWR, AMRESCO, catalog number: 97061-980 )
  28. Sodium acetate, trihydrate (Avantor Performance Materials, MACRON, catalog number: 7364-06 )
  29. Hydrochloric acid (ACS plus) (Fisher Scientific, catalog number: A144-212 )
  30. Glycerol (ACS grade) (EMD Millipore, catalog number: GX0185-5 )
  31. Transcription buffer (1x) (see Recipes)
  32. Transcription buffer (5x) (see Recipes)
  33. Transcription stop buffer (see Recipes)
  34. Tris-borate EDTA buffer (TBE) (see Recipes)
  35. TBE + 0.3 M sodium acetate (see Recipes)

Equipment

  1. NanoDrop 2000c spectrophotometer ND2000C (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000/2000c , catalog number: ND-2000C)
  2. Glass plate
  3. Block digital heater w/20 tapered hole blocks (VWR, catalog numbers: 12621-088 ; 13259-002 )
  4. 5424 table top centrifuge with w/FA-45-24-11 rotor (Eppendorf, mdoel: 5424/5424 R , catalog number: 5424000410)
  5. Powerpack HV powersupply (Bio-Rad Laboratories, model: PowerPac HV Power Supply, catalog number: 1645056 )
  6. Sequi-gen GT sequencing gel system (38 x 30 cm gel) (Bio-Rad Laboratories, catalog number: 1653862 )
    Note: This product has been discontinued.
  7. Hydrotech vacuum pump (Bio-Rad Laboratories, catalog number: 1651781 )
  8. Model 583 gel dryer (Bio-Rad Laboratories, model: Model 583, catalog number: 1651745 )
  9. DNA Engine Dyad PCR Machine 4 x 48 well blocks (Bio-Rad Laboratories)
  10. Unmounted phosphor exposure screen (35 x 43 cm) (GE Healthcare, model: General Purpose Screens, catalog number: 63-0034-79 )
  11. Storm 840 scanner (Molecular Dynamics, model: Storm 840 )
  12. Windows computer (HP, model: Compaq dc7700 )

Software

  1. Excel (Microsoft)
  2. ImageQuant (GE)
  3. SigmaPlot (Systat)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Bird, J. G., Nickels, B. E. and Ebright, R. H. (2017). RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs. Bio-protocol 7(12): e2336. DOI: 10.21769/BioProtoc.2336.

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微生物学 > 微生物生物化学 > RNA

分子生物学 > RNA > 转录

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