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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   

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

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

Notes:

  1. The presented procedure is for analysis of Escherichia coli RNAP and the transcription initiation factor σ70. The procedure can be adapted to analysis of any other RNAP by replacing E. coli RNAP with the RNAP of interest, replacing the transcription factor σ70 by the transcription initiation factor(s) used by the RNAP of interest, replacing the promoter by a promoter used by the RNAP of interest, and replacing the transcription buffer with a transcription buffer suitable for the RNAP of interest.
  2.  DNA templates are designed to facilitate generation and analysis of defined initial RNA products formed using NAD+, NADH, dpCoA, or ATP as the initiating substrate (Figure 2A). Initiation using NAD+, NADH, dpCoA, or ATP requires A:T (i.e., template-strand T) at the transcription start site (position +1; Bird et al., 2016). Use of CTP as the extending nucleotide requires C:G (i.e., template-strand G) at the first position downstream of the transcription start site (position +2). Generation of defined initial RNA products representing one, and only one, nucleotide-addition reaction requires a T:A or C:G (i.e., template-strand A or G, which are not complementary to NTPs present in reactions).


    Figure 2. Determination of relative efficiencies of transcription initiation with NAD+ vs. transcription initiation with ATP. A. dsDNA transcription templates containing RNAI and T7A1 promoters (positions -40 to +3; promoter elements and transcription start sites in gray boxes); B. Representative raw data (initial RNA products of transcription reactions performed in the presence of 1 mM NAD+, 0.01-0.5 mM ATP, and [α32P]-CTP as extending nucleotide. C and D. Relative efficiencies of transcription initiation with NAD+ vs. transcription initiation with ATP [(kcat/KM, NCIN)/(kcat/KM, ATP)]. Calculation using logarithmic regression (C; best-fit line for data points with NAD+pC/(pppApC + NAD+pC) values between 0.2 and 0.8 in (C) or non-linear regression (D); best-fit curve for data points with NAD+pC/(pppApC + NAD+pC) values between 0 and 1.

  1. Generation of transcription templates
    1. Prepare template and primers as follows:
      1. 100 nt oligodeoxyribonucleotide corresponding to promoter template-strand positions -65 to +35 (‘template’; IDT, Inc.; dissolved in water to 100 μM).
      2. 20 nt oligodeoxyribonucleotide complementary to promoter template-strand positions -65 to -46 (‘forward primer’; IDT, Inc.; dissolved in water to 100 μM).
      3. 20 nt oligodeoxyribonucleotide identical to promoter template-strand positions +16 to +35 (‘reverse primer’; IDT, Inc.; dissolved in water to 100 μM).
    2. Run PCR as follows (35 cycles):


      PCR cycles:


    3. Purify PCR products using QIAquick PCR purification kit (two columns per PCR reaction, each eluted in 40-50 μl water). (Two columns are needed because the DNA quantity exceeds the binding capacity of a single column.)
    4. Analyze aliquots by 2% agarose gel electrophoresis to confirm production of 100 bp double-stranded DNA fragment (‘dsDNA transcription template’).
    5. Quantify dsDNA transcription template by UV-Vis spectrophotometry, and adjust concentration to 0.5-1 μM in water.

  2. Transcription reactions
    Template + RNAP mix


    NCIN + NTP mixes


    Note: The water, 5x transcription buffer, [α32P]-CTP, CTP, and NCIN are pre-mixed to yield a ‘master mix’, of which 4 μl is added to 1 μl of the appropriate ATP dilution.
    1. Incubate Template + RNAP mix at 37 °C for 15 min to enable formation of a catalytically competent RNAP-promoter open complex.
    2. Add 5 μl Template + RNAP mix to pre-warmed NCIN + NTP mixes, and incubate 10 min at 37 °C.
    3. Add 10 μl ice-cold stop buffer. (Keep samples on ice if gel is to be loaded immediately, or at -20 °C if gel is to be loaded later.)
      Note: Do not boil samples that contain NCIN-capped RNA before loading gels. (NCINs can be heat-labile.)
    4. Analyze products by electrophoresis on 7.5 M urea 20% polyacrylamide nucleotide sequencing gel (National Diagnostics, Inc.; prepared per instructions of the vendor, but using one-half of specified ammonium persulfate and Temed concentrations)
      1. Pour gel.
      2. Pre-run gel at 50 W for 20-60 min before loading samples (top reservoir buffer, TBE; bottom reservoir buffer, TBE containing 0.3 M sodium acetate).
      3. Load samples (5 μl per lane).
        Optional: In a lane adjacent to sample lanes, load 1 μl transcription stop buffer with added 0.025% amaranth red as a marker. Amaranth red migrates similarly to free NTPs and allows a visual estimation of migration of dinucleotide products.
      4. Run gel at 50 W (buffers as above) for 120 min or until desired separation is achieved.
        Note: The addition of 0.3 M sodium acetate to the bottom reservoir buffer creates a salt gradient that compresses and improves resolution for short RNA products (Vo et al., 2003).
    5. Disassemble gel apparatus. Wrap gel on glass plate with polyethylene wrap, and expose to storage-phosphor screen for 3-18 h at 4 °C.

Data analysis

  1. Scan storage phosphor screen using storage phosphor imager (Storm, Typhoon, or equivalent; GE, Inc.).
  2. Open gel images in ImageQuant (GE, Inc.). and quantify band intensities using one of the following methods:
    1. Box method: Using the ImageQuant ‘Rectangle’ tool, draw uniform-size boxes around bands of interest (defining box for first band, and then copying and pasting box to each other band of interest). Perform background correction (either by using ImageQuant ‘Background Correction’ tool to define and subtract average background, or by manually subtracting background for an identically sized box placed in a region without bands). Quantify band intensities using the ImageQuant ‘Volume Report’ tool.
    2. Line method: Using the ImageQuant ‘Line’ tool, draw uniform-width lines vertically through each lane (defining line and adjusting line-width to be slightly narrower than bands for first lane, and then copying and pasting line to each other lane). Using ImageQuant ‘Create Graph’ tool, create graph reporting area under the line for each lane, define bands, and correct for background using ImageQuant ‘Peak Finder’ tool or ImageQuant ‘Define Peak’, ‘Split Peak’, and base-line-adjustment tools. Quantify band intensities using ImageQuant ‘Area Report’ tool.
  3. Calculate relative efficiencies of NCIN-mediated initiation vs. ATP-mediated initiation [(kcat/KM, NCIN)/(kcat, ATP/KM, ATP)] using one of the following methods:
    1. Logarithmic regression (using Excel or SigmaPlot): Plot observed values of NCINpC/(pppApC + NCINpC) vs. [NCIN]/[ATP] on a semi-log plot, using only observed values of NCINpC/(pppApC + NCINpC) between 0.2 and 0.8 [i.e., using only values of NCINpC/(pppApC + NCINpC) for the part of the curve that can be approximated as a line]. Perform logarithmic regression, fitting data to:

                   y = y0 + a[ln(x)]

      where, y is NCINpC/(pppApC + NCINpC), x is [NCIN]/[ATP], and y0 and a are regression parameters. The resulting fit yields the value of x for which y = 0.5. The relative efficiency (kcat/KM, NCIN)/(kcat/KM, ATP) is equal to 1/x.
    2. Non-linear regression (using SigmaPlot): Plot observed values of NCINpC/(pppApC + NCINpC) vs. [NCIN]/[ATP] on semi-log plot, using all observed values of NCINpC/(pppApC + NCINpC) [i.e., using values of NCINpC/(pppApC + NCINpC) not only for the part of the curve that can be approximated as a line but also for the parts of the curve that cannot be approximated as a line]. Perform non-linear regression, fitting data to:



      where, y is NCINpC/(pppApC + NCINpC), x is [NCIN]/[ATP], and a and b are regression parameters. The resulting fit yields the value of x for which y = 0.5. The relative efficiency (kcat/KM, NCIN)/(kcat/KM, ATP) is equal to 1/x.

Recipes

  1. Transcription buffer (1x)
    10 mM Tris HCl pH 8.0
    40 mM KCl
    10 mM MgCl2
    0.1 mM EDTA
    1 mM DTT
    0.1 mg/ml BSA
  2. Transcription buffer (5x)
    50 mM Tris HCl pH 8.0
    200 mM KCl
    50 mM MgCl2
    0.5 mM EDTA
    5 mM DTT
    0.5 mg/ml BSA
  3. Transcription stop buffer
    100 mM Tris HCl pH 8.0
    18 mM EDTA
    1.25% SDS
    90% formamide
    0.025% xylene cyanol
    0.025% bromophenol blue
    0.025% amaranth red
  4. Tris-borate EDTA buffer (TBE)
    90 mM Tris base
    90 mM boric acid
    2 mM EDTA disodium salt
  5. TBE + 0.3 M sodium acetate
    90 mM Tris base
    90 mM boric acid
    2 mM EDTA disodium salt
    300 mM sodium acetate

Acknowledgments

This work was supported by National Institutes of Health grants GM118059 (B.E.N.) and GM041376 (R.H.E.). The protocol was adapted from methods reported in Bird et al. (2016).

References

  1. Artsimovitch, I., Svetlov, V., Murakami, K. S. and Landick, R. (2003). Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions. J Biol Chem 278(14): 12344-12355.
  2. Barvik, I., Rejman, D., Panova, N., Sanderova, H. and Krasny, L. (2016). Non-canonical transcription initiation: the expanding universe of transcription initiating substrates. FEMS Microbiol Rev.
  3. Bird, J. G., Zhang, Y., Tian, Y., Panova, N., Barvik, I., Greene, L., Liu, M., Buckley, B., Krasny, L., Lee, J. K., Kaplan, C. D., Ebright, R. H. and Nickels, B. E. (2016). The mechanism of RNA 5’ capping with NAD+, NADH and desphospho-CoA. Nature 535(7612): 444-447.
  4. Ebright, R. H. (2000). RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II. J Mol Biol 304(5): 687-698.
  5. Jiao, X., Doamekpor, S. K., Bird, J. G., Nickels, B. E., Tong, L., Hart, R. P. and Kiledjian, M. (2017). 5' end nicotinamide adenine dinucleotide cap in human cells promotes RNA decay through DXO-mediated deNADding. Cell 168(6): 1015-1027 e1010.
  6. Lane, W. J. and Darst, S. A. (2010). Molecular evolution of multisubunit RNA polymerases: sequence analysis. J Mol Biol 395(4): 671-685.
  7. Marr, M. T. and Roberts, J. W. (1997). Promoter recognition as measured by binding of polymerase to nontemplate strand oligonucleotide. Science 276(5316): 1258-1260.
  8. Mukhopadhyay, J., Mekler, V., Kortkhonjia, E., Kapanidis, A. N., Ebright, Y. W. and Ebright, R. H. (2003). Fluorescence resonance energy transfer (FRET) in analysis of transcription-complex structure and function. Methods Enzymol 371: 144-159.
  9. Nickels, B. E. and Dove, S. L. (2011). NanoRNAs: a class of small RNAs that can prime transcription initiation in bacteria. J Mol Biol 412(5): 772-781.
  10. Perdue, S. A. and Roberts, J. W. (2010). A backtrack-inducing sequence is an essential component of Escherichia coli sigma(70)-dependent promoter-proximal pausing. Mol Microbiol 78(3): 636-650.
  11. Ruff, E. F., Record, M. T., Jr. and Artsimovitch, I. (2015). Initial events in bacterial transcription initiation. Biomolecules 5(2): 1035-1062.
  12. Vo, N. V., Hsu, L. M., Kane, C. M. and Chamberlin, M. J. (2003). In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 3. Influences of individual DNA elements within the promoter recognition region on abortive initiation and promoter escape. Biochemistry 42(13): 3798-3811.
  13. Walters, R. W., Matheny, T., Mizoue, L. S., Rao, B. S., Muhlrad, D. and Parker, R. (2017). Identification of NAD+ capped mRNAs in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 114(3): 480-485.
  14. Winkelman, J. T., Vvedenskaya, I. O., Zhang, Y., Zhang, Y., Bird, J. G., Taylor, D. M., Gourse, R. L., Ebright, R. H. and Nickels, B. E. (2016). Multiplexed protein-DNA cross-linking: Scrunching in transcription start site selection. Science 351(6277): 1090-1093.

简介

最近已经确定,含有腺嘌呤的辅因子,包括烟酰胺腺嘌呤二核苷酸(NAD +),还原型烟酰胺腺嘌呤二核苷酸(NADH)和3'-脱磷酸辅酶A(dpCoA)可以作为“非规范起始核苷酸” NCIN),用于通过细菌和真核细胞RNA聚合酶(RNAP)进行转录起始,并且通过启动子序列确定反应的效率(Bird等,2016)。 在这里,我们描述了使用NCIN与使用三磷酸核苷(NTP)对于给定启动子序列的转录起始来定量转录起始的相对效率的方案。
【背景】在细菌,古细菌和真核生物中的转录由序列,结构和机制保守的多亚基RNA聚合酶(RNAPs)进行(Ebright,2000; Lane和Darst,2010)。为了启动转录,RNAP与一个或多个引发因子一起结合称为“启动子”的特异性DNA序列,并解开启动子DNA以形成含有未解链“转录泡”的RNAP启动子开放复合物(RPo)(图1A; Ruff等人,2015)。 RNAP然后通过扩增(“剔除”)或收缩(“抗锯齿”)转录起始点来选择转录起始位点,以将转录起始位点的核苷酸置于RNAP活性中心起始位点(“i位点”)和扩增位点'i + 1位点')结合i位点的互补起始核苷酸底物和“i + 1”位点的互补延伸底物,并催化磷酸二酯键形成产生初始RNA产物(Winkelman等, 2016)。
在标准的从头转录启动中,起始底物是核苷三磷酸(NTP),通常为ATP或GTP(Nickels and Dove,2011)。然而,最近已经确定,含有腺嘌呤的辅因子,包括烟酰胺腺嘌呤二核苷酸(NAD +),还原型烟酰胺腺嘌呤二核苷酸(NADH)和3'-脱磷酸辅酶A(dpCoA)可用作替代的起始底物(NCAN),产生具有独特的5'端结构,稳定性和翻译效率的NCIN-封端RNA产物(图1B-1C; Bird等,2016; Barvik等,2016; Jiao et al。,2017; Walters et al。,2017)。进一步证实,NCIN介导的启动与NTP介导的起始的相对效率由启动子序列确定(Bird等,2016)。
在这里,我们描述了一个方案,用于确定给定启动子序列的NCIN介导的转录起始与ATP介导的转录起始(kcat / KM,NCIN)/(kcat / KM,ATP)的相对效率。该方案涉及在具有恒定浓度的NCIN和不同浓度的ATP的一组转录反应中产生放射性标记的初始RNA产物,然后定量NCIN起始的RNA和总RNA,随后绘制观察到的NCIN起始RNA与总RNA的比率作为NCIN浓度与ATP浓度比的函数。
转录启动 A. RNAP启动子开放复合物(RPo)与解开的转录气泡。 灰色RNAP; 蓝色,-10元素核苷酸; i和i + 1,RNAP活性中心引发核苷酸结合位点和延伸核苷酸结合位点; 盒,DNA核苷酸(模板链核苷酸上方的非模板链核苷酸)。 B. ATP和NAD +,红,ATP和NAD +中相同原子的结构; C.使用ATP(上)或转录起始使用NAD +(底部)在转录起始中形成的初始RNA产物。 左侧子屏幕显示在i站点启动ATP或NAD +结合; 正确的子面板显示使用CTP形成的起始RNA产物作为延伸核苷酸。 ATP,NAD +和CTP的红盒,腺苷和胞嘧啶部分; 绿色盒子,NAD +的烟酰胺 - 核糖体部分。

关键字:RNA聚合酶, 转录, 非典型起始核苷酸(NCIN), RNA加帽, 从头开始RNA加帽, NAD+, NADH, 3'-去磷酸辅酶A

材料和试剂

  1. 电子。大肠杆菌RNA聚合酶 70全酶
    注意:如Mukhopadhyay等人所述。 (2003)或购买(New England Biolabs,目录号:M0551S)
  2. 电子。大肠杆菌RNA聚合酶核心酶
    注意:如Artimovitch等人所做的那样。 (2003)。
  3. 电子。大肠杆菌σ 70
    注:准备为Marr和Roberts(1997); Perdue和Roberts(2010)。
  4. (I级,游离酸)(Roche Molecular Systems,目录号:10127965001)
  5. NADH(I级,游离酸)(Roche Molecular Systems,目录号:10107735001)
  6. Phusion Flash HF主混合物(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:F548L)
    注意:用于生成转录模板。
  7. 寡脱氧核糖核苷酸(模板和引物)(Integrated DNA Technologies, www.IDTdna.com
  8. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
  9. TEMED(Avantor Performance Materials,J.T.Baker ®,目录号:4098-01)
  10. 过硫酸铵(VWR,AMRESCO,目录号:97064-594)
  11. GeneMate LE快速溶解琼脂糖(BioExpress,目录号:E-3119-500)
  12. 3'-去磷酸酶A(Sigma-Aldrich,目录号:D3385)
  13. SequaGel测序系统(国家诊断,目录号:EC-833)
  14. 高纯度rNTP组(ATP,UTP,GTP,CTP)(100mM)(GE Healthcare,目录号:27-2025-01)
  15. [A-32] PTP-CTP EasyTide(3,000Ci/mmol)(250μCi)(Perkin Elmer,目录号:BLU508H250UC)
  16. Tris碱(VWR,AMRESCO,目录号:97061-800)
  17. 氯化钾(KCl)(EMD Millipore,目录号:PX1405-1)
  18. 六水合氯化镁(EMD Millipore,目录号:5980-500GM)
  19. EDTA二钠盐水合物(1kg)(VWR,AMRESCO,目录号:97061-018)
  20. 二硫苏糖醇(DTT)(Gold Bio,目录号:DTT50)
  21. 牛血清白蛋白(BSA)级分V(Alfa Aesar,Affymetrix/USB,目录号:J10857)
  22. 十二烷基硫酸钠(SDS)(VWR,AMRESCO,目录号:97064-470)
  23. 去离子甲酰胺(EMD Millipore,目录号:4610-100ML)
  24. 二甲苯胞苷(Sigma-Aldrich,目录号:X4126-10G)
  25. 溴苯酚蓝(EMD Millipore,目录号:BX1410-7)
  26. 苋菜红(Acros Organics,目录号:AC15303-0250)
  27. 硼酸(ACS级)(VWR,AMRESCO,目录号:97061-980)
  28. 乙酸钠,三水合物(Avantor Performance Materials,MACRON,目录号:7364-06)
  29. 盐酸(ACS plus)(Fisher Scientific,目录号:A144-212)
  30. 甘油(ACS级)(EMD Millipore,目录号:GX0185-5)
  31. 转录缓冲液(1x)(见配方)
  32. 转录缓冲液(5x)(见配方)
  33. 转录停止缓冲液(见配方)
  34. 三硼酸盐EDTA缓冲液(TBE)(参见食谱)
  35. TBE + 0.3M醋酸钠(参见食谱)

设备

  1. NanoDrop 2000c分光光度计ND2000C(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000/2000c,目录号:ND-2000C)
  2. 玻璃板
  3. 块数字加热器带有20个锥形孔块(VWR,目录号:12621-088; 13259-002)
  4. 5424台式离心机,带有FA-45-24-11转子(Eppendorf,mdoel:5424/5424 R,目录号:5424000410)
  5. Powerpack HV电源(Bio-Rad实验室,型号:PowerPac HV电源,目录号:1645056)
  6. SequiGGGG测序凝胶系统(38×30cm凝胶)(Bio-Rad Laboratories,目录号:1653862)
    注意:本产品已停产。
  7. Hydrotech真空泵(Bio-Rad Laboratories,目录号:1651781)
  8. 583型凝胶干燥器(Bio-Rad Laboratories,型号:Model 583,目录号:1651745)
  9. DNA引擎染色体PCR机4×48孔块(Bio-Rad Laboratories)
  10. 未安装的荧光体曝光屏(35×43厘米)(GE Healthcare,型号:通用屏幕,目录号:63-0034-79)
  11. Storm 840扫描仪(分子动力学,型号:Storm 840)
  12. Windows计算机(HP,型号:Compaq dc7700)

软件

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

程序

注意:

  1. 所述方法用于分析大肠杆菌RNAP和转录起始因子σ 70。该方法可以适用于任何其它RNAP的分析,用目标RNAP替换大肠杆菌RNAP,用感兴趣的RNAP所使用的转录起始因子代替转录因子σ 70 ,用目标RNAP所使用的启动子取代启动子,用适合感兴趣的RNAP的转录缓冲液代替转录缓冲液。
  2. DNA模板设计用于促进使用NAD ,NADH,dpCoA或ATP形成的定义的初始RNA产物的产生和分析作为引发底物(图2A)。使用NAD + ,NADH,dpCoA或ATP的起始在转录起始位点(位置+1; Bird等人,2016)需要A:T(即模板链T)。使用CTP作为延伸核苷酸需要在转录起始位点(位置+2)下游的第一个位置使用C:G(即模板链G)。产生代表一个且仅一个核苷酸加成反应的定义的初始RNA产物需要T:A或C:G(即与反应中存在的NTP不互补的模板链A或G)。 >


    图2.用NAD确定转录起始的相对效率 + vs。包含RNAI和T7A1启动子(位置-40至+3;灰盒中的启动子元件和转录起始位点)的dsDNA转录模板; B.代表性原始数据(在1mM NAD +,0.01-0.5mM ATP和[α 32 P] -CTP的存在下进行的转录反应的初始RNA产物作为扩增核苷酸C和D.与ATP转录起始的相对效率(ATP )与ATP的转录起始[(k /K> ,NCIN)/(k /K> M,ATP)]使用对数回归计算(C;用NAD (C)或非线性回归(D)中的0.2和0.8之间的平均值/(pppApC + NAD + pC)值;用NAD < pC /(pppApC + NAD + pC)值介于0和1之间。

  1. 生成转录模板
    1. 准备模板和底漆如下:
      1. 对应于启动子模板链位置-65至+35("模板"; IDT,Inc。;溶于水至100μM)的100nt寡脱氧核糖核苷酸。
      2. 与启动子模板链位置-65至-46("正向引物"; IDT,Inc。;溶于水至100μM)互补的20nt寡脱氧核糖核苷酸。
      3. 与启动子模板链位置相同的20nt寡脱氧核糖核苷酸+16至+35("反向引物"; IDT,Inc。;溶于水至100μM)。
    2. 运行PCR如下(35个循环):


      PCR循环:


    3. 使用QIAquick PCR纯化试剂盒纯化PCR产物(每个PCR反应两个柱,每个在40-50μl水中洗脱)。 (需要两列,因为DNA量超过单柱的结合能力。)
    4. 通过2%琼脂糖凝胶电泳分析等分试样,以确认100 bp双链DNA片段('dsDNA转录模板')的产生。
    5. 通过紫外 - 可见分光光度法测定dsDNA转录模板,并在水中调节浓度至0.5-1μM
  2. 转录反应
    模板+ RNAP组合


    NCIN + NTP混合


    注意:将水,5x转录缓冲液[α]/P> -CTP,CTP和NCIN预先混合至产生"主混合物",其中4μl加入到1μl适当的ATP稀释液中
    1. 在37℃下孵育模板+ RNAP混合物15分钟,以形成催化能力的RNAP启动子开放复合物。
    2. 将5μlTemplate + RNAP混合物加入预热的NCIN + NTP混合物中,37℃孵育10分钟。
    3. 加入10μl冰冷的停止缓冲液。 (如果要立即加载凝胶,请将样品保存在冰上,如果要稍后加载凝胶,则保存在-20°C。)
      注意:在加载凝胶之前,不要煮含含有NCIN封端RNA的样品。 (NCIN可以是热不稳定的。)
    4. 通过7.5M尿素20%聚丙烯酰胺核苷酸测序凝胶电泳分析产物(National Diagnostics,Inc。;根据供应商的说明制备,但使用一半的特定过硫酸铵和Temed浓度)
      1. 倒凝胶。
      2. 预装凝胶在50 W下加载样品(顶部储存缓冲液,TBE;底部储存缓冲液,含0.3M乙酸钠的TBE)20-60分钟。
      3. 加载样品(每泳道5μl) 可选:在与样品通道相邻的通道中,加入1μl转录停止缓冲液,加入0.025%苋菜红作为标记。苋菜红类似地迁移到免费的NTPs,并允许视觉估计二核苷酸产物的迁移
      4. 以50W(如上所述的缓冲液)运行凝胶120分钟或达到理想的分离 注意:向底部储存器缓冲液中加入0.3M乙酸钠产生盐梯度,其压缩并提高短RNA产物的分辨率(Vo等人,2003)。
    5. 拆解凝胶装置。在玻璃板上用聚乙烯包裹包裹凝胶,并在4℃下暴露于储存荧光屏3-18小时

数据分析

  1. 使用存储荧光成像仪(Storm,Typhoon或等同物; GE,Inc.)扫描存储荧光屏。
  2. 在ImageQuant(GE,Inc.)中打开凝胶图像。并使用以下方法之一量化带强度:
    1. 框方法:使用ImageQuant'Rectangle'工具,在感兴趣的频段(在第一个频段的定义框,然后复制和粘贴框到每个其他感兴趣的频段)中绘制统一大小的框。执行背景校正(通过使用ImageQuant'背景校正'工具来定义和减去平均背景,或通过手动减去放置在没有波段的区域中的相同大小的框的背景)。使用ImageQuant'Volume Report'工具量化频带强度。
    2. 线方法:使用ImageQuant'Line'工具,通过每个通道垂直绘制均匀宽度的线(定义线和调整线宽比第一个通道的带略窄,然后将线复制并粘贴到每个通道)。使用ImageQuant"创建图形"工具,在每个通道的行下创建图形报告区域,使用ImageQuant'Peak Finder'工具或ImageQuant'Define Peak','Split Peak'和base-line-调整工具使用ImageQuant'区域报告'工具量化频带强度。
  3. 计算NCIN介导的起始与ATP介导的起始的相对效率(NCIN)/(k cat) ATP/K ATP)],使用以下方法之一:
    1. 对数回归(使用Excel或SigmaPlot):在半对数图上绘制NCINpC /(pppApC + NCINpC)对[NCIN]/[ATP]的观察值,仅使用观察到的NCINpC /(pppApC + NCINpC)值在0.2和0.8 [ ie ,仅使用可以近似为行的部分的NCINpC /(pppApC + NCINpC)的值]。执行对数回归,拟合数据到:

                    y = y + a [ln( x )]

      其中, y 是NCINpC /(pppApC + NCINpC), x 是[NCIN]/[ATP],而 y > 0 和 a 是回归参数。所得到的拟合产生 x = 0.5的值。 ATP的相对效率(kN)/N m N,NCIN)/(k cat/N/K M)等于1/x 。
    2. 非线性回归(使用SigmaPlot):使用NCINpC /(pppApC + NCINpC)的所有观察值,在半对数图上绘制NCINpC /(pppApC + NCINpC)与[NCIN]/[ATP]的观察值的比较[ > ie ,使用NCINpC /(pppApC + NCINpC)的值,不仅可以将曲线中可以近似为线的部分,也可以用于不能近似为线的曲线部分]。执行非线性回归,拟合数据到:



      其中, y 是NCINpC /(pppApC + NCINpC), x 是[NCIN]/[ATP],而 a 和 是回归参数。所得到的拟合产生 x = 0.5的值。 ATP的相对效率(kN)/N m N,NCIN)/(k cat/N/K M)等于1/x 。

食谱

  1. 转录缓冲液(1x)
    10 mM Tris HCl pH 8.0
    40 mM KCl
    10mM MgCl 2
    0.1 mM EDTA
    1 mM DTT
    0.1 mg/ml BSA
  2. 转录缓冲液(5x)
    50 mM Tris HCl pH 8.0
    200 mM KCl
    50mM MgCl 2
    0.5 mM EDTA
    5 mM DTT
    0.5 mg/ml BSA
  3. 转录停止缓冲区
    100mM Tris HCl pH 8.0
    18 mM EDTA
    1.25%SDS
    90%甲酰胺
    0.025%二甲苯氰胺
    0.025%溴酚蓝
    0.025%苋菜红
  4. 三硼酸盐EDTA缓冲液(TBE)
    90 mM Tris碱基
    90 mM硼酸
    2mM EDTA二钠盐
  5. TBE + 0.3M醋酸钠
    90 mM Tris碱基
    90 mM硼酸
    2mM EDTA二钠盐
    300 mM醋酸钠

致谢

这项工作得到了美国国家卫生研究院授予GM118059(B.E.N.)和GM041376(R.H.E.)的支持。该方案适用于Bird等人报道的方法(2016)。

参考

  1. Artsimovitch,I.,Svetlov,V.,Murakami,KS和Landick,R。(2003)。  大肠杆菌共同超表达RNA聚合酶亚基允许分离和分析缺乏谱系特异性序列插入的突变酶。 J Biol Chem 278(14):12344-12355。
  2. Barvik,I.,Rejman,D.,Panova,N.,Sanderova,H.and Krasny,L.(2016)。  非典型转录启动:转录启动底物的扩展宇宙。 FEMS Microbiol Rev 。
  3. Bird,JG,Zhang,Y.,Tian,Y.,Panova,N.,Barvik,I.,Greene,L.,Liu,M.,Buckley,B.,Krasny,L.,Lee,JK,Kaplan, CD,Ebright,RH和Nickels,BE(2016)。  使用NAD ,NADH和desphospho-CoA的RNA 5'封端的机制。自然界535(7612):444-447。
  4. Ebright,RH(2000)。  RNA polymerase:结构相似性细菌RNA聚合酶和真核RNA聚合酶II之间。分子生物 304(5):687-698。
  5. Jiao,X.,Doamekpor,SK,Bird,JG,Nickels,BE,Tong,L.,Hart,RP and Kiledjian,M.(2017)。  5'末端烟酰胺腺嘌呤二核苷酸帽在人类细胞中通过DXO介导的脱氨酸促进RNA衰减。/168(6):1015-1027 e1010。
  6. Lane,WJ and Darst,SA(2010)。  分子多亚基RNA聚合酶的进化:序列分析。分子生物 395(4):671-685。
  7. Marr,M.T。和Roberts,J.W。(1997)。 通过聚合酶与非模板链寡核苷酸结合测量的启动子识别。 科学 276(5316):1258-1260。
  8. Mukhopadhyay,J.,Mekler,V.,Kortkhonjia,E.,Kapanidis,AN,Ebright,YW和Ebright,RH(2003)。< a class ="ke-insertfile"href ="http: ncbi.nlm.nih.gov/pubmed/14712697"target ="_ blank">荧光共振能量转移(FRET)在转录 - 复合物结构和功能分析中的应用方法Enzymol 371: 144-159。
  9. Nickels,BE and Dove,SL(2011)。  NanoRNAs :一类可以在细菌中引发转录起始的小RNA。 J Mol Biol 412(5):772-781。
  10. Perdue,SA和Roberts,JW(2010)。  A回溯诱导序列是大肠杆菌(70)依赖性启动子近端暂停的重要组成部分.Mol Microbiol 78(3):636-650 。
  11. Ruff,EF,Record,MT,Jr. and Artsimovitch,I.(2015)。  细菌转录启动中的初始事件。生物分子 5(2):1035-1062。
  12. Vo,NV,Hsu,LM,Kane,CM和Chamberlin,MJ(2003)。 通过大肠杆菌RNA聚合酶进行的转录启动研究。 3.启动子识别区域内单个DNA元件对流产起始和启动子逃逸的影响。生物化学 42(13):3798-3811。
  13. Walters,RW,Matheny,T.,Mizoue,LS,Rao,BS,Muhlrad,D.and Parker,R。(2017)。< a class ="ke-insertfile"href ="http: ncbi.nlm.nih.gov/pubmed/28031484"target ="_ blank">在酿酒酵母中鉴定NAD + 加帽的mRNA 。 Natl Acad Sci USA 114(3):480-485。
  14. Winkelman,JT,Vvedenskaya,IO,Zhang,Y.,Zhang,Y.,Bird,JG,Taylor,DM,Gourse,RL,Ebright,RH and Nickels,BE(2016)。< a class =插入文件"href ="http://www.ncbi.nlm.nih.gov/pubmed/26941320"target ="_ blank">复用蛋白质-DNA交联:在转录起始位点选择中清除。 >科学 351(6277):1090-1093。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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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