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Aug 2019

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Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates
体外磷酸化的RNA聚合酶II羧基末端域底物的电泳迁移率测定   

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Abstract

Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques exist to identify and quantify the degree of CTD phosphorylation, including phosphorylation-specific antibodies and mass spectrometry. Electrophoretic mobility shift assays (EMSAs) have been utilized since the discovery of CTD phosphorylation and continue to represent a simple, direct, and widely applicable approach for qualitatively monitoring CTD phosphorylation. We present a standardized method for EMSA analysis of recombinant GST-CTD substrates phosphorylated by a variety of CTD kinases. Strategies to analyze samples under both denatured/reduced and semi-native conditions are provided. This method represents a simple, direct, and reproducible means to monitor CTD phosphorylation in recombinant substrates utilizing equipment common to molecular biology labs and readily applicable to downstream analyses including immunoblotting and mass spectrometry.

Keywords: RNA polymerase II (RNA聚合酶II), Transcription (转录), Phosphorylation (磷酸化), Kinase (激酶), mRNA (mRNA), CTD Code (羧基末端结构域代码)

Background

Eukaryotic RNA polymerase II (RNAPII) generates all protein-coding mRNAs, small nuclear, small nucleolar, and many micro RNAs (Jeronimo et al., 2013; Mayfield et al., 2016). A variety of mechanisms regulate RNAPII activity to confer specificity to gene expression and facilitate biological processes. Among these is the direct post-translational modification of RNAPII itself in the form of phosphorylation (Mayfield et al., 2016), prolyl isomerization (Mayfield et al., 2015), methylation (Dias et al., 2015), and acetylation (Schroder et al., 2013). Some of the best-studied modifications are phosphorylations of the C-terminal domain of RNAPII’s largest subunit RPB1 (CTD). This domain is evolutionarily conserved from yeast to mammals and composed of a species-specific number of repeats of the consensus amino acid heptad YSPTSPS (conventionally numbered as Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, and Ser7). Phosphorylations of specific heptad residues act to recruit transcription factors and coincide with distinct stages of the transcription cycle. For instance, Ser5 phosphorylation hallmarks transcription initiation while Ser2 phosphorylation is apparent during the transition from promoter escape to productive elongation. These dynamic post-translational modifications and precisely recruited protein factors constitute the ‘CTD Code’ for eukaryotic transcription and ensure the production of mature and functional transcripts (Jeronimo et al., 2013; Mayfield et al., 2016).

RNAPII is recruited to the pre-initiation complex when the CTD is in the unphosphorylated form. Upon transcription initiation, the CTD becomes hyperphosphorylated, indicative of a transcriptionally engaged RNAPII. At transcription termination, the CTD undergoes dephosphorylation and is recycled to initiate another round of transcription. This is achieved through the action of multiple CTD kinases; including CDK7, of the TFIIH complex, and CDK9, of the Positive transcription elongation factor b (P-TEFb) complex; and their counterparts the CTD phosphatases, including SSU72 and CTDP1 (Jeronimo et al., 2013; Mayfield et al., 2016). Distinct pools of unphosphorylated and hyperphosphorylated RPB1 are detected in cell lysates using polyacrylamide gel electrophoresis (PAGE) due to a dramatic shift in the isoelectric point of RPB1 resulting from hyperphosphorylation. This physical characteristic of hyperphosphorylated RPB1 allowed for the initial discovery and characterization of the CTD and continues to be a useful tool in the study of CTD phosphorylation (Corden et al., 1985; Mayfield et al., 2019).

There are many ways to interrogate both the identity and abundance of modifications in CTD substrates, including antibodies (Jeronimo et al., 2013), mass spectrometry (Mayfield et al., 2017) and biophysical approaches like small-angle x-ray scattering (Portz et al., 2017). However, EMSA has the advantage of direct visualization, easy setup, and a rapid completion time without the requirement for specialized equipment. Here we treat GST-CTD fusion proteins with various CTD kinases, resolve reaction products in electrophoresis, and visualize product bands to describe reaction outcomes qualitatively. This approach is useful for verifying kinase and phosphatase activity against CTD substrates, processive/stochastic addition or removal of phosphates, and qualitative estimation of the number of phosphates added to CTD substrates. This approach is informative under both denatured/reduced and semi-native conditions allowing for tunable resolution and increasing downstream applicability in techniques like immunoblotting and band-excision coupled to mass spectrometry analysis.

Materials and Reagents

  1. SnakeSkin Dialysis Tubing, 10K MWCO, 22 mm (Thermo Scientific, catalog number: 68100 )
  2. Amicon Ultra-15 Centrifugal Filter Unit (Millipore Sigma, catalog number: UFC900308 )
  3. GST-yeast CTD Bacterial Expression Vector (subcloned as in Mayfield et al., 2019, or similar)
  4. BL21 (DE3) Competent Cells (Thermo Scientific, catalog number: EC0114 )
  5. Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
  6. Sodium chloride (Sigma-Aldrich, catalog number: S9888 )
  7. Bacto tryptone (Gibco, catalog number: 211699 )
  8. Bacto agar (Fisher Scientific, catalog number: DF0140010 )
  9. Kanamycin monosulfate (GoldBio, catalog number: K1205 )
  10. IPTG (GoldBio, catalog number: 367931 )
  11. Tris Base (Fisher Scientific, catalog number: BP1525 )
  12. Hydrochloric acid (Fisher Chemical, catalog number: A144SI212 )
  13. Sodium Hydroxide (Fisher Chemical, catalog number: S3201 )
  14. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  15. Glycerol (Fisher Chemical, catalog number: G334 )
  16. Imidazole (Sigma-Aldrich, catalog number: 56750 )
  17. β-mercaptoethanol (Acros Organics, catalog number: 125472500 )
  18. Pierce Coomassie Plus (Bradford) Assay Reagent (Thermo Scientific, catalog number: 23238 )
  19. Ni-NTA His Bind Resin (EMD Millipore, catalog number: 70666 )
  20. Magnesium chloride hexahydrate (Sigma-Aldrich, catalog number M9272)
  21. Adenosine 5’ triphosphate disodium salt hydrate (Sigma-Aldrich, catalog number: A26209 )
  22. Cdk9/Cyclin T1 Protein, active (Millipore, catalog number: 14-685 )
  23. Cdk7/Cyclin H/MAT1 (CAK complex) Protein, active (Millipore, catalog number: 14476 )
  24. c-Abl kinase (ProQinase, catalog number: 0992-0000-1 )
  25. pET-28 a vector (Novagen, catalog number: 698643 )
  26. Sodium Dodecyl Sulfate (SDS) (OmniPur, catalog number: 7910 )
  27. Bromophenol blue (Sigma-Aldrich, catalog number: B55255G )
  28. 30% Acrylamide/Bis Solution 37.5:1 (Bio-Rad, catalog number: 1610158 )
  29. Ammonium persulfate (APS) (Fisher Scientific, Research Products International Corp, catalog number: A2050010.0 )
  30. N,N,N’,N’-Tetramethyl ethylenediamine TEMED (Fisher Scientific, catalog number: BP15020 )
  31. Glycine (Fisher Scientific, catalog number: BP381-5 )
  32. PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Scientific, catalog number 26619), or equivalent pre-stained protein ladder
  33. Brilliant Blue R250 (Sigma, catalog number: B0149 )
  34. Ethanol, Ethyl alcohol 200 Proof (Pharmaco, catalog number: 111000200 )
  35. Glacial Acetic acid (Fisher Chemical, catalog number: A38C212 )
  36. LB Medium (1 L) (see Recipes)
  37. 1,000x Kanamycin stock (50 mg/ml) (see Recipes)
  38. 1,000x IPTG stock (400 mM) (see Recipes)
  39. Lysis buffer (see Recipes)
  40. Wash buffer (see Recipes)
  41. Elution buffer (see Recipes)
  42. Dialysis buffer (see Recipes)
  43. 4x Kinase Buffer (see Recipes)
  44. 4x ATP (5 ml) (see Recipes)
  45. 4x GST-yeast CTD substrate (see Recipes)
  46. 4x P-TEFb kinase solution (see Recipes)
  47. 4x TFIIH kinase solution (see Recipes)
  48. 4x c-Abl kinase solution (see Recipes)
  49. 1 M Tris-HCl pH 6.8 (500 ml) (see Recipes)
  50. 1.5 M Tris-HCl pH 8.8 (500 ml) (see Recipes)
  51. 10% SDS (100 ml) (see Recipes)
  52. 2x Laemmli’s sample buffer (see Recipes)
  53. Resolving Semi-native PAGE/SDS-PAGE Gel (10% Acrylamide; 5 ml–sufficient for 1 gel, scale as necessary) (see Recipes)
  54. Stacking Semi-native PAGE/SDS-PAGE Gel (5% Acrylamide, 2 ml–sufficient for 1 gel, scale as necessary) (see Recipes)
  55. 1x Laemmli running buffer (Tris-glycine) (1 L) (see Recipes)
  56. 1x Native Laemmli running buffer (Tris-glycine) (1 L) (see Recipes)
  57. Coomassie brilliant blue stain (100 ml) (see Recipes)
  58. Destain (see Recipes)

Equipment

  1. 1 L flasks
  2. Pipettes
  3. -80 °C freezer
  4. Bacterial Culture Incubator (Thermo Fisher, catalog number: SHKE435HP ) sufficient to hold 1 L culture volumes
  5. UV-Vis Spectrophotometer (SmartSpec Plus Spectrophotometer (Bio-Rad, catalog number: 170-2525 , or equivalent)
  6. High-Speed Floor Centrifuge with appropriate rotors for 1 L and 50 ml centrifuge containers (Thermo Scientific, model: Sorvall RC 6+ , or equivalent)
  7. Sonicator for Bacterial Lysis (Q500 Sonicator) (Qsonica, catalog number: Q500-110 , or equivalent)
  8. Econo-Column Chromatography Column 2.5 x 10 cm (Bio-Rad, catalog number: 7374251 )
  9. FPLC System with automated fraction collector and UV280 monitoring capabilities (Bio-Rad, NGC system, or equivalent)
  10. HiLoad 16/600 Superdex 200 pg (GE Lifesciences, catalog number: 28989335 , or equivalent)
  11. NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific, catalog number: ND-ONE-W , or equivalent)
  12. Mini-PROTEAN Tetra Vertical Electrophoresis cell (Bio-Rad, catalog number: 1658004 )
  13. Mini-PROTEAN Tetra cell casting stand and clamps (Bio-Rad, catalog number: 1658050 )
  14. Mini-PROTEAN Spacer Plates with 1.0 mm Integrated spacers (Bio-Rad, catalog number: 1653311 )
  15. Mini-PROTEAN Short Plates (Bio-Rad, catalog number: 1653308 )
  16. Mini-PROTEAN Comb, 15-well, 1 mm, 26 µl (Bio-Rad, catalog number: 1653360 )
  17. PowerPac Basic Power Supply (Bio-Rad, catalog number: 1645050 )
  18. G: BOX imaging systems (Syngene), or equivalent

Software

  1. ImageJ (Open source image processing software, imagej.net)

Procedure

  1. Expression and Purification of GST-yeast CTD Substrate
    Note: GST fusions of the CTDs from various organisms, including Homo sapiens, Drosophila melanogaster, and Saccharomyces cerevisiae, are utilized extensively. The easy availability of synthetic constructs containing defined numbers of heptad repeats greatly simplified cloning. The EMSA method presented here applies to all of these various substrates. However, a universal purification method does not apply to all constructs. We provide a purification protocol for a 6X-HIS tagged GST and Saccharomyces cerevisiae CTD fusions due to their historical precedence, the content of primarily consensus heptad repeats, and extensive use as a model substrate regardless of CTD modifying enzyme origin. Purification schemes for alternative CTD constructs are available throughout published literature and/or should be determined empirically.

    1. Subclone GST-yeast CTD sequence (as described in Mayfield et al., 2019) into the pET- 28 a (+) vector, or equivalent, using any standard cloning method.
    2. Transform sequence-verified plasmid into BL21 (DE3) Competent E. coli cells using conventional methods and select for transformants by growing overnight at 37 °C on an agar plate with 50 μg/ml kanamycin.
    3. Inoculate a single colony into 10ml of LB media containing 50 μg/ml kanamycin (10 ml LB + 10 μl 1,000x kanamycin stock) and grow overnight at 37 °C to generate a saturated culture.
    4. Inoculate two 1 L flasks of LB containing 50 μg/ml kanamycin (1 L LB + 1,000 μl 1,000x kanamycin stock) with 4 ml of the saturated overnight culture each flask. Incubate at 37 °C with shaking at 180 rpm. Monitor OD600 using UV-Vis spectrophotometry until the cultures reach a value of approximately 0.6-0.8.
    5. Induce expression by adding 1 ml of 1,000x IPTG stock and allow the culture to grow at 37 °C with shaking at 180 rpm for an additional four hours.
    6. Harvest cell pellet by transferring culture to appropriate 1 L centrifuge flasks, counterbalancing the flasks if necessary, and centrifuging at 5,000 x g for 20 min at room temperature. Cell pellets can be processed immediately or frozen at -20 °C for up to 1 year.
    7. Combine cell pellets from 2 L of growth media and resuspend in 100 ml of Lysis Buffer. Allow pellet to resuspend fully by stirring solution vigorously on ice for 30 min.
    8. Lyse cells via sonication using a properly tuned instrument. Sonicate suspended cells on ice using 10 cycles of 30 s continuous sonication followed by 1 min of recovery with constant stirring. In the end, the solution will appear yellowish for an effective sonication.
    9. Clear lysate of cell debris by splitting lysate equally among 50 ml centrifuge tubes, verifying they are of equal mass, and centrifuging samples at 10,000 x g for 40 min at 4 °C. Pool supernatant fractions and discard cell debris containing pellets.
    10. Apply 5 ml of Ni-NTA His Bind Resin slurry to an empty chromatography column and allow supernatant to flow through. Equilibrate beads with 5 ml of lysis buffer and combine bead/lysis buffer slurry with pooled supernatant fractions. Stir the supernatant with the beads on a stir plate in the cold room for 30 min to ensure protein binding.
    11. Apply supernatant/bead mixture to the chromatography column and allow the supernatant to flow through. Collect flow-through and pour it back over the beads one time. Your protein is now bound to the beads.
    12. Wash the beads with 100 ml of wash buffer, allowing it to flow through.
    13. Elute GST-yeast CTD from the column by adding 10 ml of Elution buffer and allowing the solution to slowly drip from the chromatography column and collect it in a clean 50 ml conical tube.
    14. Verify protein content by mixing 200 μl Pierce Coomassie Plus (Bradford) Assay Reagent with 2 μl of the elution. If protein is present, the solution turns a vibrant blue. Verify protein content and identity using classical SDS-PAGE analysis of the elution. GST-yeast CTD (as described in Mayfield et al., 2019) has a theoretical molecular weight of 48.7 kDa.
    15. Transfer eluted protein to SnakeSkin Dialysis tubing and dialyzed against 1 L of Dialysis Buffer overnight at 4 °C.
    16. Equilibrate HiLoad 16/600 Superdex 200 pg column with Dialysis Buffer using an FPLC by washing the column with 1.5 column volumes at a flow rate of 1 ml/min.
    17. Prepare an Amicon Ultra-15 Centrifugal Filter Unit by adding 5-10 ml deionized water to the unit and spinning the unit at 3,500 x g for 5 min. Discard remaining water on both sides of the molecular weight cut off filter.
    18. Apply dialyzed protein to the prepared centrifugal filter unit and centrifuge the unit at 3,500 x g for 10-min intervals at 4 °C until the volume above the molecular weight cut-off filter reaches 1 ml. Transfer the volume above the filter to a 1.5 ml tube and centrifuge at 13,000 x g at 4 °C for 10 min to remove any protein aggregates. Transfer cleared supernatant to a fresh 1.5 ml tube.
    19. Inject concentrated sample onto the FPLC and prepared column. Monitor elution absorbance at 280 nm and collect 1 ml fractions beginning at the void volume and ending at 1.2 column volumes. Using the absorbance trace, determine fractions containing GST-yeast CTD and prepare SDS-PAGE samples by mixing 20 μl of the fraction with 20 μl of 2x Laemmli’s Sample Buffer and boiling the samples at 95 °C for 5 min. Verify protein identity and purity using established SDS-PAGE and Coomassie Brilliant Blue staining protocols, similar to those presented below.
    20. Pool fractions containing GST-yeast CTD and concentrate in Amicon centrifugal unit until approximately 10mg/ml as determined by absorbance at 280 nm on NanoDrop. Aliquot to 50 μl fractions to 1.5 ml tubes and flash freeze in liquid nitrogen. Store at -80 °C.

  2. In vitro phosphorylation of GST-yeast CTD Substrate using CTD Kinases
    1. Kinase reactions
      Combine stock solutions as follows:
      5 μl 4x Kinase Buffer (Recipe 8)
      5 μl 4x GST-yeast CTD Substrate (Recipe 10)
      5 μl 4x Kinase Solution (Recipes 11, 12, 13 as desired)
      5 μl 4x ATP
      Initiate reactions by adding 4x ATP. Incubate reactions in a thermocycler at 30 °C for the desired amount of time. Reaction time to completion is kinase-dependent and should be determined empirically. In our hands, TFIIH and P-TEFb reactions approach completion after approximately 1 h, while c-Abl reactions require approximately 4 h to reach completion. Exact reaction times depend on the kinase, experimental goal, and production lot and should be determined empirically. Completed reactions can be analyzed immediately or stored at -80 °C for up to 1 year.
      Note: To date, various CTD kinases are commercially available or described in the literature. Here, we provide a reaction condition for studying CTD phosphorylation by three commercially available human CTD kinases/kinase complexes: TFIIH, P-TEFb, and c-Abl. Alternative kinases can also be used, but reaction conditions should be optimized for the kinase of interest. Special attention should be paid to the lot number, and commercial sources of kinases as their individual activity varies widely and require optimization.
    2. No kinase control reactions
      Combine stock solutions as follows:
      5 μl 4x Kinase Buffer (Recipe 8)
      5 μl 4x GST-yeast CTD Substrate (Recipe 10)
      5 μl Deionized water
      5 μl 4x ATP
      Initiate reactions by adding 4x ATP. Incubate reactions in thermocycler at 30 °C for desired amount of time. No kinase control reactions should be run in parallel with the kinase-containing reactions. Completed reactions can be analyzed immediately or stored at -80 °C for up to 1 year.

  3. Preparation of Electrophoresis Samples
    Note: Samples for both SDS-PAGE and Semi-Native PAGE are prepared identically. The denaturation/reduction of Semi-Native PAGE samples in Laemmli sample buffer increases the sharpness and resolution of final bands relative to purely native samples. It tends to increase the degree of electrophoretic mobility shift between unphosphorylated and phosphorylated substrates over that observed in SDS-PAGE.

    Quench reactions by adding 20 μl of 2x Laemmli’s Sample Buffer and boiling at 95 °C for 5 min. This yields approximately 40 μl of the final sample with a GST-yeast CTD substrate concentration of around 0.5 μg/μl.

  4. SDS-PAGE (Denaturing/Reducing)
    Note: 10% acrylamide SDS-PAGE gels are suggested here as they resolve proteins well in the mass range of GST-yeast CTD substrates. Consider alternative acrylamide concentrations when using alternative substrates or a different resolution is desired.
    1. Set up the Bio-Rad Mini-PROTEAN Tetra Cell according to factory directions with SDS- PAGE gels (Recipes 18-19) and 1x Laemmli’s Running Buffer (Tris-glycine) (Recipe 20). Load 2 μl (1 μg) of prepared electrophoresis samples to each well, making sure to include both kinase containing and no kinase control reactions. Include a molecular weight ladder.
    2. Run the gel at 150 V (constant) for approximately 1 h, or until the dye front reaches the bottom of the gel.
    3. Remove the gel from the electrophoresis chamber and glass casting and proceed immediately to Coomassie Brilliant Blue Staining or alternative downstream application.

  5. Semi-Native PAGE
    Note: 10% acrylamide Semi-native PAGE gels are suggested here as they resolve proteins well in the mass range of GST-yeast CTD substrates. Consider alternative acrylamide concentrations when using alternative substrates or a different resolution is desired.
    1. Set up the Bio-Rad Mini-PROTEAN Tetra Cell according to factory directions with Semi- native PAGE gels (Recipes 18-19) and 1x Native Laemmli’s Running Buffer (Tris-glycine) (Recipe 21). Load 2 μl (1 μg) of prepared electrophoresis samples to each well, making sure to include both kinase containing and no kinase control reactions. Include a pre-stained molecular weight ladder. The ladder masses are not directly interpretable in Semi-native PAGE but aid in reproducibility of Semi-native PAGE experiments and provide gel progress information if the proteins of interest are not well separated before the dye front leaves the gel.
    2. Run the gel at 150 V (constant) for approximately 1-4 h with an ice pack. Long runs may be necessary to obtain well-separated bands, and this time should be optimized for your particular samples.
    3. Remove the gel from the electrophoresis chamber and glass casting and proceed immediately to Coomassie Brilliant Blue Staining or alternative downstream application.

  6. Coomassie brilliant blue staining
    1. Briefly rinse gel with deionized water. Decant deionized water and add sufficient Coomassie Brilliant Blue stain to cover the gel. Incubate gel at room temperature with gentle agitation for a minimum of 1 h or overnight. Decant Coomassie Brilliant Blue stain into an appropriate container. The stain can be reused multiple times.
    2. Rinse gel thoroughly in deionized water to remove residual Coomassie Brilliant Blue stain and cover gel completely with Destain. Incubate gel at room temperature with gentle agitation until stained protein bands become visible, and background is minimal, changing the destain as necessary.

Data analysis

The destained gel can be immediately visually interpreted. In SDS-PAGE applications, phosphorylation decreases the mobility of the GST-yeast CTD substrate. This is evidenced by a higher apparent molecular weight of the kinase treated sample relative to the no kinase control sample. In the semi-native PAGE, phosphorylation increases the mobility of the GST-yeast CTD substrate. This will appear as bands of lower apparent molecular weight in the kinase treated sample relative to the no kinase control. Gels can be imaged in any conventional gel imagine system, including Chemi-Doc (Bio-Rad) and G: Box (Syngene) systems. Cropping of images can be performed in ImageJ. ImageJ may also be used to adjust brightness and contrast equally across the image to increase the interpretability of the gels. Special care should be taken to ensure image modifications are applied judiciously and equivalently across all samples considered.
  EMSA analyses have been employed throughout the literature. Examples of the method presented here can be found in Mayfield et al. (2019). An application of the SDS-PAGE EMSA to two types of GST-CTD substrate can be found in Figures 1C and 2A. Semi-native PAGE is presented as a useful alternative when increase resolution is desired and requires minimal alteration of the more established SDS-PAGE protocol as in Supplementary Figure 1D.

Recipes

  1. LB Medium (1 L)
    Combine the following in 900 ml deionized water:
    10 g Bacto tryptone
    5 g yeast extract
    10 g NaCl
    12 g Bacto agar (for agar plates only)
    1. Shake or stir the solution to dissolve solutes
    2. Adjust final volume to 1 L using deionized water
    3. Sterilize by autoclaving for 20 min at 15 psi on a liquid cycle
    4. If desired, add antibiotic once media has cooled completely (for liquid media) or is molten but comfortable to the touch (for agar plates). Plates should then be poured under aseptic conditions
  2. 1,000x Kanamycin stock (50 mg/ml)
    1. Dissolve 500 mg of kanamycin monosulfate (M.W. = 582.60) in 9 ml deionized water
    2. Adjust volume to 10 ml with deionized water
    3. Filter sterilize solution through a 0.22 μm filter and aliquot into sterile tubes
    4. Store at -20 °C
  3. 1,000x IPTG stock (400 mM)
    1. Dissolve 950 mg of Isopropyl-beta-D-thiogalactoside (IPTG, M.W. = 238.30) in 9 ml deionized water
    2. Adjust volume to 10 ml with deionized water
    3. Filter sterilize solution through a 0.22 μm filter and aliquot into sterile tubes
    4. Store at -20 °C
  4. Lysis buffer (1 L)
    50 mM Tris-HCl pH 8.0 (50 ml diluted from 1 M Tris-His pH 8.0 stock [Recipe 14])
    500 mM NaCl (29.25 g)
    15 mM Imidazole (15 ml from 1 M stock)
    10% Glycerol (10 ml from 100% stock)
    0.1% Triton X-100 (1 ml from 100% stock)
    10 mM β-mercaptoethanol (0.7 ml from stock of 14.3 M)
  5. Wash buffer (1 L)
    50 mM Tris-HCl pH 8.0 (50 ml diluted from 1 M Tris-His pH 8.0 stock [Recipe 14])
    500 mM NaCl (29.25 g)
    15 mM Imidazole (15 ml from 1 M stock)
    10 mM β-mercaptoethanol (0.7 ml from stock of 14.3 M)
  6. Elution buffer (1 L)
    50 mM Tris-HCl pH 8.0 (50 ml diluted from 1 M Tris-His pH 8.0 stock [Recipe 14])
    500 mM NaCl (29.25 g)
    400 mM Imidazole (400 ml from 1 M stock)
    10 mM β-mercaptoethanol (0.7 ml from stock of 14.3 M)
  7. Dialysis buffer (1 L)
    50 mM Tris-HCl pH 8.0 (50 ml diluted from 1 M Tris-His pH 8.0 stock [Recipe 14])
    50 mM NaCl (2.9 g)
    10 mM β-mercaptoethanol (0.7 ml from the stock of 14.3 M)
    For FPLC applications, the buffer should be filtered through a 0.4 μm filter to remove any particulates
  8. 4x Kinase Buffer (100 ml)
    200 mM Tris-HCl pH 7.5 (20 ml diluted from 1 M Tris-His pH 7.5 stock [Recipe 14])
    200 mM MgCl2 (10 ml from 2 M stock)
  9. 4x ATP (5 ml)
    22 mg Adenosine 5’ triphosphate disodium salt hydrate
    1 M Tris-HCl pH 7.5 (variable)
    1. Dissolve adenosine 5’ triphosphate disodium salt hydrate in 4 ml of deionized water
    2. Verify pH by spotting a small amount (1-2 μl) of solution onto pH paper. The initial solution should be very acidic
    3. Adjust pH with 1 M Tris-HCl pH 7.5 by adding 20-100 μl at a time and checking the pH after each addition. Once the pH registers 7.5, adjust the final volume of the solution to 5 ml using deionized water. Adjusting the pH of the ATP solution is essential
  10. 10% SDS (100 ml)
    10 g Sodium Dodecyl Sulfate (SDS)
    80 ml Deionized water
  11. 2x Laemmli’s Sample Buffer
    0.125 M Tris-HCl pH 6.8
    4% SDS (w/v)
    10% β-mercaptoethanol (v/v)
    20% Glycerol (v/v)
    0.02% Bromophenol blue (w/v)
  12. Resolving Semi-native PAGE/SDS-PAGE Gel (10% Acrylamide; 5 ml–sufficient for 1 gel, scale as necessary)
    1.7 ml 30% Acrylamide
    1.3 ml 1.5M Tris-HCl pH 8.8
    1.9 ml Deionized water
    50 μl 10% SDS (w/v) (for SDS-PAGE gels only)
    50 μl Deionized water (for Semi-native PAGE gels only)
    50 μl 10% Ammonium persulfate (w/v)
    4 μl TEMED
  13. Stacking Semi-native PAGE/SDS-PAGE Gel (5% Acrylamide, 2 ml–sufficient for 1 gel, scale as necessary)
    330 μl 30% Acrylamide
    250 μl 1M Tris-HCl pH 6.8
    1.4 ml Deionized water
    20 μl 10% SDS (w/v) (for SDS-PAGE gels only)
    20 μl Deionized water (for Semi-Native PAGE gels only)
    20 μl 10% Ammonium persulfate (w/v)
    2 μl TEMED
  14. 1x Laemmli running buffer (Tris-glycine) (1 L)
    Combine the following in 900 ml deionized water:
    3.03 g Tris base
    14.2 g Glycine
    1 ml 10% SDS (w/v)
    Allow all reagents to dissolve and adjust pH to 8.3 if necessary. Adjust volume to 1 L with additional deionized water.
    1. Adjust pH to 6.8 (or 7.5 and 8.0) using concentrated hydrochloric acid and bring the final volume to 500 ml with additional deionized water
    2. Store at room temperature
  15. 1x Native Laemmli running buffer (Tris-glycine) (1 L)
    Combine the following in 900 ml deionized water:
    3.03 g Tris base
    14.2 g Glycine
    Allow all reagents to dissolve and adjust pH to 8.3 if necessary. Adjust volume to 1 L with additional deionized water.
    1. Combine Tris base and deionized water
    2. Adjust pH to 8.8 with concentrated hydrochloric acid and bring the final volume to 500 ml with additional deionized water
    3. Store at room temperature
  16. Coomassie Brilliant Blue Stain (100 ml)
    Combine the following:
    0.25 g Coomassie Brilliant Blue R250
    45 ml Ethanol
    45 ml Deionized water
    10 ml Glacial acetic acid
    Stir constantly until completely dissolved
    1. Combine SDS and deionized water and stir until dissolved
    2. Adjust final volume to 100 ml with additional deionized water
    3. Store at room temperature
  17. Destain
    Combine the following:
    50 ml Ethanol
    75 ml Glacial acetic acid
    875 ml Deionized water
    Stir or invert container until thoroughly mixed
  18. Resolving Semi-native PAGE/SDS-PAGE Gel (10% Acrylamide; 5 ml–sufficient for 1 gel, scale as necessary)
    1.7 ml 30% Acrylamide
    1.3 ml 1.5M Tris-HCl pH 8.8
    1.9 ml Deionized water
    50 μl 10% SDS (w/v) (for SDS-PAGE gels only)
    50 μl Deionized water (for Semi-native PAGE gels only)
    50 μl 10% Ammonium persulfate (w/v)
    4 μl TEMED
    1. Combine 30% Acrylamide, 1.5 M Tris-HCl pH 8.8, deionized water, SDS (for SDS-PAGE), or additional deionized water (for Semi-native PAGE) in a screw-top vial and invert to combine
    2. Add ammonium persulfate and TEMED to initiate polymerization, invert gently, and transfer solution to prepared Bio-Rad Mini-PROTEAN gel casting
    3. Fill casting, leaving sufficient space at the top of gel for stacking layer and comb
    4. Gently apply a layer of 100% ethanol to the top of resolving gel and allow it to polymerize completely and pour off ethanol
  19. Stacking Semi-native PAGE/SDS-PAGE Gel (5% Acrylamide, 2 ml–sufficient for 1 gel, scale as necessary)
    330 μl 30% Acrylamide
    250 μl 1M Tris-HCl pH 6.8
    1.4 ml Deionized water
    20 μl 10% SDS (w/v) (for SDS-PAGE gels only)
    20 μl Deionized water (for Semi-Native PAGE gels only)
    20 μl 10% Ammonium persulfate (w/v)
    2 μl TEMED
    1. Combine 30% Acrylamide, 1 M Tris-HCl pH 6.8, deionized water, and SDS (for SDS-PAGE) or additional deionized water (for Semi-native PAGE) in a screw-top vial and invert to combine
    2. Add ammonium persulfate and TEMED to initiate polymerization, invert gently, and transfer solution to prepared Bio-Rad Mini-PROTEAN gel casting containing polymerized resolving gel
    3. Fill the remainder of the casting with stacking gel solution and insert a comb
    4. Allow the gel to polymerize completely
    5. Store final gels wrapped in moist paper towels and plastic wrap at 4 °C for up to 2 weeks
  20. 1x Laemmli running buffer (Tris-glycine) (1 L)
    Combine the following in 900 ml deionized water:
    3.03 g Tris base
    14.2 g Glycine
    1 ml 10% SDS (w/v)
    Allow all reagents to dissolve and adjust pH to 8.3 if necessary. Adjust volume to 1 L with additional deionized water.
  21. 1x Native Laemmli running buffer (Tris-glycine) (1 L)
    Combine the following in 900 ml deionized water:
    3.03 g Tris base
    14.2 g Glycine
    Allow all reagents to dissolve and adjust pH to 8.3 if necessary. Adjust volume to 1 L with additional deionized water.
  22. Coomassie Brilliant Blue Stain (100 ml)
    Combine the following:
    0.25 g Coomassie Brilliant Blue R250
    45 ml Ethanol
    45 ml Deionized water
    10 ml Glacial acetic acid
    Stir constantly until completely dissolved
  23. Destain
    Combine the following:
    50 ml Ethanol
    75 ml Glacial acetic acid
    875 ml Deionized water
    Stir or invert container until thoroughly mixed

Acknowledgments

This work is supported by grants from the National Institutes of Health (R01 GM104896 and 125882 to YJZ) and Welch Foundation (F-1778 to YJZ). Methods presented here are adapted from previous work (Mayfield et al., 2019).

Competing interests

The authors declare no competing financial interests.

References

  1. Corden, J. L., Cadena, D. L., Ahearn, J. M., Jr. and Dahmus, M. E. (1985). A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II. Proc Natl Acad Sci U S A 82(23): 7934-7938. 
  2. Dias, J. D., Rito, T., Torlai Triglia, E., Kukalev, A., Ferrai, C., Chotalia, M., Brookes, E., Kimura, H. and Pombo, A. (2015). Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells. Elife 4: 11215. 
  3. Jeronimo, C., Bataille, A. R. and Robert, F. (2013). The writers, readers, and functions of the RNA polymerase II C-terminal domain code. Chem Rev 113(11): 8491-8522.
  4. Mayfield, J. E., Burkholder, N. T. and Zhang, Y. J. (2016). Dephosphorylating eukaryotic RNA polymerase II. Biochim Biophys Acta 1864(4): 372-387. 
  5. Mayfield, J. E., Fan, S., Wei, S., Zhang, M., Li, B., Ellington, A. D., Etzkorn, F. A. and Zhang, Y. J. (2015). Chemical tools to decipher regulation of phosphatases by proline isomerization on eukaryotic RNA Polymerase II. ACS Chem Biol 10(10): 2405-2414. 
  6. Mayfield, J. E., Irani, S., Escobar, E. E., Zhang, Z., Burkholder, N. T., Robinson, M. R., Mehaffey, M. R., Sipe, S. N., Yang, W., Prescott, N. A., Kathuria, K. R., Liu, Z., Brodbelt, J. S. and Zhang, Y. (2019). Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb. Elife 8: 48725. 
  7. Mayfield, J. E., Robinson, M. R., Cotham, V. C., Irani, S., Matthews, W. L., Ram, A., Gilmour, D. S., Cannon, J. R., Zhang, Y. J. and Brodbelt, J. S. (2017). Mapping the phosphorylation pattern of Drosophila melanogaster RNA polymerase II carboxyl-terminal domain using ultraviolet photodissociation mass spectrometry. ACS Chem Biol 12(1): 153-162.
  8. Portz, B., Lu, F., Gibbs, E. B., Mayfield, J. E., Rachel Mehaffey, M., Zhang, Y. J., Brodbelt, J. S., Showalter, S. A. and Gilmour, D. S. (2017). Structural heterogeneity in the intrinsically disordered RNA polymerase II C-terminal domain. Nat Commun 8: 15231.
  9. Schroder, S., Herker, E., Itzen, F., He, D., Thomas, S., Gilchrist, D. A., Kaehlcke, K., Cho, S., Pollard, K. S., Capra, J. A., Schnolzer, M., Cole, P. A., Geyer, M., Bruneau, B. G., Adelman, K. and Ott, M. (2013). Acetylation of RNA polymerase II regulates growth-factor-induced gene transcription in mammalian cells. Mol Cell 52(3): 314-324.

简介

[摘要 ] 真核RNA聚合酶II转录所有编码蛋白质的mRNA,并且受到高度调节。指导RNA聚合酶II并促进mRNA的共转录加工的关键机制是其高度重复的羧基末端结构域(CTD)的磷酸化。最大的亚基RPB1位于特定残基。存在多种鉴定和定量CTD磷酸化程度的技术,包括磷酸化特异性抗体和质谱法。自发现CTD磷酸化和本文提出了一种标准化的方法,用于EMSA分析被多种CTD激酶磷酸化的重组GST-CTD底物的EMSA方法,以及在变性/还原和还原条件下分析样品的策略。提供了半本地条件。此方法表示简单,直接,以及使用分子生物学实验室通用的设备监测重组底物中CTD磷酸化的可重现方法,该设备可轻松应用于下游分析,包括免疫印迹和质谱分析。

[背景 ] 真核生物RNA聚合酶II(RNAPII)产生所有蛋白质编码的mRNA,小核,小核仁,和许多微小RNA (杰罗尼莫等,2013;梅菲尔德。等,2016) 。各种机制中规范RNAPII活动要赋予特异性基因表达和促进生物处理工艺。在这些是直接翻译后修饰中RNAPII自己在形式的磷酸化(梅菲尔德等,2016) ,脯氨酰异构(梅菲尔德等,2015) ,甲基化(迪亚斯等人,2015年)和乙酰化(交银施罗德等,2013) 。一些研究最多的修饰是磷酸化的C端结构域RNAPII最大的亚基RPB1中(CTD) ,该结构域进化保守从酵母到并合成哺乳动物物种特异性重复次数共有氨基酸七重复YSPTSPS(常规编号为Tyr1,SER2,Pro3的,THR4,Ser5,PRO6和Ser7)中磷酸化的具体七重复残留法招收陈德良 例如,Ser5磷酸化标志着转录的启动,而Ser2磷酸化在从启动子逃逸到有效延伸的过程中很明显,这些动态的翻译后修饰和精确募集的蛋白质因子构成了CTD。真核转录的代码”,并确保产生成熟和功能性的转录本(Jeronimo 等,2013; Mayfield 等,2016)。

当CTD处于未磷酸化形式时,RNAPII被募集到起始前复合体中;在转录起始时,CTD变得超磷酸化,表明转录参与了RNAPII。在转录终止时,CTD进行了去磷酸化,并被再循环以启动另一轮转录是通过多种CTD激酶(包括TFIIH复合物的CDK7和正转录延伸因子b(P- TEFb )复合物;以及与之对应的CTD磷酸酶,包括SSU72和CTDP1 (Jeronimo 等人,2013; Mayfield 等人,2016)。由于过度磷酸化导致RPB1的等电点发生明显变化,因此使用聚丙烯酰胺凝胶电泳(PAGE)在细胞裂解物中检测到了未磷酸化和超磷酸化的RPB1的不同库。磷酸化的RPB1的物理特性可用于CTD的初步发现和表征 并且仍然是研究CTD磷酸化的有用工具(Corden 等,1985; Mayfield 等,2019)。

有很多方法可以查询CTD底物中修饰的同一性和丰富性,包括抗体(Jeronimo 等,2013),质谱(Mayfield 等,2017)和生物物理方法,例如小角度X射线散射( Portz et al。,2017)。然而,EMSA的优点是直接可视化,易于设置,完成时间短而无需专用设备。在这里,我们用各种CTD激酶处理GST-CTD融合蛋白,分离出反应产物电泳,并可视化产物带以定性描述反应结果。此方法可用于验证针对CTD底物的激酶和磷酸酶活性,进行性/随机添加或去除磷酸盐以及定性估计添加到CTD底物中的磷酸盐数量。在变性/还原和半原生条件下均可提供信息,从而可调整分辨率并增加下游应用 免疫印迹和条带切除技术结合质谱分析。

关键字:RNA聚合酶II, 转录, 磷酸化, 激酶, mRNA, 羧基末端结构域代码

材料和试剂


 


SnakeSkin 透析管,10K MWCO,22毫米(Thermo Scientific,目录号:68100)
Amicon Ultra-15离心过滤器(Millipore Sigma,目录号:UFC900308)
GST酵母CTD细菌表达载体(亚克隆于Mayfield 等人,2019 ,或类似的结果)
BL21(DE3)感受态细胞(Thermo Scientific,目录号:EC0114)
酵母ë XTRACT(Sigma-Aldrich公司,目录号:Y1625)
氯化钠(Sigma-Aldrich,目录号:S9888)
Bacto t ryptone(Gibco,目录号:211699)
细菌培养用一个噶尔(FIS 她的科学,目录号:DF0140010)
卡那霉素单硫酸盐(GoldBio ,目录号:K1205)
IPTG(GoldBio ,目录号:367931)
Tris Base(Fisher Scientific,目录号:BP1525)
盐酸(Fisher Chemical,目录号:A144SI212)
氢氧化钠(Fisher Chemical,目录号:S3201)
海卫一X-100(Sigma-Aldrich,目录号:X100)
甘油(Fis 她的化学药品,目录号:G334)
咪唑(Sigma-Aldrich,目录号:56750)
β - 巯基乙醇(ACROS 有机物,目录号:125472500)
Pierce Coomassie Plus(Bradford)分析试剂(Thermo Scientific,目录号:23238)
Ni-NTA His Bind Resin(EMD密理博,目录号:70666)
六水合氯化镁(Sigma-Aldrich,目录号M9272)
腺苷5'三磷酸二钠盐水合物(Sigma-Aldrich,目录号:A26209)
活性Cdk9 / Cyclin T1蛋白(Millipore,目录号:14-685)
活性Cdk7 / Cyclin H / MAT1(CAK复合物)(Millipore,目录号:14476)
c- Abl 激酶(ProQinase ,目录号:0992-0000-1)
pET-28载体(Novagen ,目录号698643)
十二烷基硫酸钠(SDS)(OmniPur ,目录号:7910)
溴酚蓝(Sigma - Aldrich,目录号:B55255G)
30%丙烯酰胺/双解溶液37.5:1(Bio - R ad,目录号:1610158)
过硫酸铵(APS)(Fis 她的Scientific,Research Products International Corp,目录号:A2050010.0)
N,N,N',N'-四氢甲基乙二胺TEMED(Fis her Scientific,目录号:BP15020)
甘氨酸(适应她的科学,目录号:BP381-5)
PageRuler 加上预染蛋白质梯,10至250 kDa的物(Thermo Scientific,目录号26619),或等同的预染色的蛋白质梯
艳蓝R250(Sigma,目录号:B0149)
乙醇,乙醇200证明(Pharmaco ,目录号:111000200)
冰醋酸(Fis her Chemical,目录号:A38C212)
LB中(1 L)(请参阅食谱)
1,000x卡那霉素原液(50 mg / ml)(请参阅食谱)
1,000x IPTG库存(400 mM)(请参阅食谱)
裂解缓冲液(请参见食谱)
清洗缓冲液(请参阅配方)
洗脱缓冲液(请参见配方)
透析缓冲液(请参见配方)
4x激酶缓冲液(请参阅食谱)
4倍ATP(5毫升)(请参阅食谱)
4x GST- 酵母CTD底物(请参阅食谱)
4x P- TEFb 激酶溶液(请参阅食谱)
4x TFIIH激酶溶液(请参阅食谱)
4x c- Abl 激酶溶液(请参阅食谱)
1 M Tris-HCl pH 6.8(500 ml)(请参阅食谱)
1.5 M Tris-HCl pH 8.8(500 ml)(请参阅食谱)
10%SDS(100 ml)(请参阅食谱)
2 倍Laemmli的样本缓冲区(请参阅食谱)
分离半天然PAGE / SDS- PAGE凝胶(10%丙烯酰胺; 5 ml – 足够用于1凝胶,必要时可缩放)(请参见食谱)
叠加式半天然PAGE / SD S-PAGE凝胶(5%丙烯酰胺,2 ml – 足以用于1凝胶,必要时可缩放)(请参见食谱)
1个Laemmli 运行缓冲液(Tris-甘氨酸)(1 L)(请参阅食谱)
1个本机Laemmli 运行缓冲液(Tris-甘氨酸)(1 L)(请参阅食谱)
考马斯亮蓝色染料(100毫升)(请参阅食谱)
脱色(请参阅食谱)
 


设备


 


1升烧瓶
移液器
-80°C冷冻室
细菌培养箱(Thermo Fisher,目录号:SHKE435HP)足以容纳1 L培养体积
可见分光光度计-紫外线(SmartSpec Plus分光光度计(Bio -R Ad,目录号:170-2525或等效产品)
带有适用于1 L和50 ml离心容器的合适转子的高速落地离心机(Thermo Scientific,型号:Sorvall RC 6+,或同等产品)
超声波仪为细菌裂解(Q500 超声波仪)(Qsonica ,目录号:Q500-110,或等同物)
经济柱色谱柱2.5 x 10厘米(Bio - Rad ,目录号:7374251)
FPLC系统的自动级分收集器和UV280监控功能(生物- Rad公司,NGC系统,或等同物)
HiLoad 16/600 Superdex 200 pg (GE Lifesciences,目录号:28989335或同等产品)
Nano D Rop 一微体积紫外可见分光光度计(Thermo Scientific,目录号:ND.-ONE-W或等效产品)
Mini-PROTEAN Tetra垂直电泳池(Bio - Rad ,目录号1658004)
Mini-PROTEAN Tetra细胞浇铸支架和夹具(Bio - Rad ,目录号:1658050)
带1.0 mm集成垫片的Mini-PROTEAN垫片(Bio - Rad ,产品目录号:1653311)
Mini-PROTEAN短板(Bio - Rad ,目录号:1653308)
Mini-PROTEAN梳子,15孔,1 mm,26 µl(Bio - Rad ,目录号:1653360)
PowerPac 基本电源(Bio - Rad ,目录号:1645050)
G:BOX成像系统(Syngene )或同等水平
 


软件


 


ImageJ(开源图像处理软件,imagej.net)
 


程序


 


GST酵母CTD 底物的表达与纯化
注意:广泛利用了来自不同生物体(包括智人,果蝇和酿酒酵母)的CTD的GST融合体,包含定义数量的七足动物重复序列的合成构建体的易得性大大简化了克隆。此处介绍的EMSA方法适用于所有但是,通用的纯化方法并不适用于所有构建体。由于其历史悠久的历史,主要共有的七肽重复序列的含量以及我们提供的6X-HIS标记的GST和酿酒酵母CTD融合的纯化方案。不论CTD修饰酶的来源如何,均可广泛用作模型底物。替代的CTD构建体的纯化方案可在整个已发表的文献中找到和/或应凭经验确定。


 


亚克隆GST-CTD的酵母序列(如描述梅菲尔德等人,2019 进入)的pET - 28(+)载体,或等价的,使用任何标准克隆方法。             
使用常规方法将经过序列验证的质粒转化为BL21(DE3)感受态大肠杆菌细胞,并通过在含有50μg / ml 卡那霉素的琼脂平板上于37 °C 下生长过夜来选择转化体。             
接种单个菌落到含有50 LB培养基10ml的微克/ ml卡那霉素(10毫升LB + 10 微升1,000X卡那霉素母液)和在37℃下生长过夜以产生饱和培养。              
含有LB的接种2个1升烧瓶中50 微克/ ml卡那霉素(1L LB + 1000 微升1,000X 卡那霉素母液)用4ml饱和过夜培养各flask.Incubate的在37℃下以180 rpm.Monitor摇动OD 600 使用紫外可见分光光度法,直到培养物达到约0.6-0.8的值。                           
通过加入1ml的1诱导表达,000 X IPTG库存,并允许培养物以在37℃下生长在180 rpm振摇额外4小时。             
通过将培养物转移到合适的1 L离心瓶中收获细胞沉淀,必要时将其平衡,然后在室温下以5,000 xg离心20分钟。细胞沉淀可以立即处理或在-20°C下冷冻长达1年。             
合并来自2 L生长培养基的细胞沉淀,并重悬于100 ml的裂解缓冲液中,通过在冰上剧烈搅拌溶液30分钟,使沉淀完全重悬。
通过使用适当调整的仪器进行超声处理来裂解细胞。使用30次连续声处理的10个循环对冰上的悬浮细胞进行超声处理,然后在持续搅拌下恢复1分钟,最后溶液将呈现微黄色,从而可以进行有效的超声处理。
通过将裂解液在50 ml离心管中等分,确认裂解液的质量,并在4 °C下以10,000 xg离心40分钟,以清除细胞碎片的裂解液,合并液滴部分并丢弃含有沉淀的细胞碎片。
将5 ml Ni-NTA His Bind Resin浆液倒入空色谱柱中,使液体流过,用5 ml裂解缓冲液平衡微珠,并将微珠/裂解缓冲液与合并的上清液馏分合并。在冷室中搅拌30分钟以确保蛋白质结合。             
将流通液/流通液混合物加到色谱柱上,让流通液通过,收集流通液,然后倒回珠子一次,此时您的蛋白质便与珠粒结合了。
用100毫升洗涤缓冲液洗涤珠子,使其流过。
加入10 ml洗脱缓冲液,使溶液从色谱柱中缓慢滴下,从色谱柱上洗脱GST-酵母CTD ,并将其收集在干净的50 ml锥形管中。
检查蛋白质含量,由混合200个Myueru 皮尔斯考加(布拉德福德)测定试剂用2 Myueru 的溶出。如果蛋白存在,溶液变成一个充满活力的蓝色。检查蛋白质含量和标识采用经典的SDS-PAGE分析洗脱。GST -酵母CTD (如Mayfield 等人所述,2019)具有48.7 kDa 的理论分子量。
将诱导的蛋白质转移到SnakeSkin 透析管中,并在1 °C 的透析缓冲液中于4°C 透析过夜。             
使用FPLC,通过用1.5倍柱体积的柱以1 ml / min的流速洗涤柱,用透析缓冲液平衡HiLoad 16/600 Superdex 200 pg 柱。
通过向设备中加入5-10 ml去离子水并以3500 xg 的转速旋转设备5分钟来制备Amicon Ultra-15离心过滤器,并丢弃分子量截留过滤器两侧的剩余水。
将透析的蛋白质涂到准备好的离心过滤器上,并在3500 xg下于4°C下以10分钟的间隔离心,直到截留分子量的过滤器上方的体积达到1 ml,然后将过滤器上方的体积转移至1.5 ml试管,在4°C下以13,000 xg离心10分钟以去除任何蛋白质聚集体,然后转移至新的1.5 ml试管中。                                         
将浓缩的样品注入FPLC和准备好的色谱柱中,在280nm处监测洗脱吸光度并收集1 ml从空体积开始至1.2柱体积的级分使用吸光度示踪确定含有GST-酵母C TD的级分并制备SDS-PAGE通过混合20份样品微升的馏分用20 微升2的X 的Laemmli的样品缓冲液并在95℃下沸腾样品5分钟。验证蛋白质身份和纯度使用建立的SDS-PAGE和考马斯亮蓝染色的协议,类似于那些呈现下面。                                         
收集包含Ng GST-酵母CTD的馏分,并在Amicon 离心装置中浓缩至约10 mg / Ml(通过在纳米D Rop上在280 Nm处的吸光度确定)。分装至50 Myueru ;分装到1.5 Ml的试管中,并在液氮中速冻-- 80℃。             
 


使用CTD激酶对GST-酵母CTD 底物进行体外磷酸化
激酶反应
              合并库存解决方案,如下所示:


                            5 微升4X激酶缓冲液(配方8)


                            5 微升4倍GST-CTD酵母底物(配方10)


                            5 微升4X激酶溶液(如所期望配方11,12,13)


                            5 微升4x ATP


              通过在30℃下加入4倍ATP.Incubate反应在热循环仪用于期望发起反应的时间。反应完成时间量是激酶依赖性和应empirically.In我们手中来确定,TFIIH和P- TEFB 反应接近完成后大约1小时,而c- Abl 反应大约需要4小时才能完成。确切的反应时间取决于激酶,实验目标和生产量,应凭经验确定。完成的反应可立即进行分析或在-80°C储存长达一年。                           


              注意,迄今为止,各种CTD激酶是可商购的或在文献中进行了描述。在此,我们提供了通过三种可商购的人CTD激酶/激酶复合物TFIIH,P - TEFb 和c-Abl 研究CTD磷酸化的反应条件。也可以使用其他激酶,但应针对感兴趣的激酶优化反应条件,应特别注意激酶的批号和商业来源,因为它们的个体活性差异很大且需要优化。             


              无激酶控制反应
              合并库存解决方案,如下所示:


                            5 微升4X激酶缓冲液(配方8)


                            5 微升4倍GST-CTD酵母底物(配方10)


                            5 微升去离子水


                            5 微升4x ATP


              加入4倍ATP来引发反应。在30°C的热循环仪中孵育所需的时间。不应将激酶控制反应与含激酶的反应并行进行。已完成的反应应立即进行分析或保存在-80°C长达一年。


 


电泳样品的制备
注意:SDS-PAGE和Semi-Native PAGE样品的制备方法完全相同.Laemmli 样品缓冲液中Semi-Native PAGE样品的变性/还原相对于纯天然样品,增加了最终条带的清晰度和分辨率,这往往会增加磷酸迁移率在未磷酸化和磷酸化底物之间的迁移程度超过了在SDS-PAGE中观察到的程度。


 


加入20骤冷反应微升2×的莱城mmli的样品缓冲液并在95℃下煮沸5个min.This 产率约40微升与最终样品的GST-CTD酵母的大约0.5底物浓度微克/ 微升。             


 


SDS-PAGE(变性/还原)
注意:建议使用10%丙烯酰胺SDS-PAGE凝胶,因为它们在GST酵母CTD 底物的质量范围内能很好地分辨蛋白质。使用替代底物或不同分辨率时,请考虑替代丙烯酰胺的浓度。


根据与SDS-工厂方向设置的Bio-Rad公司的Mini-PROTEAN利细胞PAGE凝胶(配方18-19)和1x 的Laemmli的运行缓冲液(Tris-甘氨酸)(配方20)。负载2 微升(1 微克的制备)对每个孔进行电泳,确保同时包含激酶和不进行激酶对照反应,包括分子量阶梯。                           
在150V(恒定)下运行凝胶约1小时,或直到染料前沿到达凝胶底部。
从电泳室和玻璃铸件中取出凝胶,并立即进行考马斯亮蓝染色或其他下游应用。             
 


半本地PAGE
注意:建议使用10%丙烯酰胺半天然PAGE凝胶,因为它们可以在GST酵母CTD 底物的质量范围内很好地分离蛋白质。使用替代底物或不同分辨率时,请考虑替代丙烯酰胺的浓度。


根据工厂说明,使用半天然PAGE凝胶(配方18-19)和1 x 天然Laemmli的运行缓冲液(Tris-甘氨酸)(配方21)按照出厂说明设置Bio-Rad Mini-PROTEAN Tetra Cell。负载2μl (1μg )制备的电泳样品到每个孔中,确保同时包含激酶和不进行激酶控制反应。包括预先染色的分子量梯子。梯子质量不能在Semi-native PAGE中直接解释,但有助于Semi的重现性-天然PAGE实验,如果感兴趣的蛋白质在染料前沿离开凝胶之前未得到很好的分离,则可提供凝胶进展信息。                                         
用冰袋在150 V(恒定)下运行凝胶约1-4小时,可能需要长时间运行才能获得分离良好的条带,这次应针对您的特定样品进行优化。
从电泳室和玻璃铸件中取出凝胶,并立即进行考马斯亮蓝染色或其他下游应用。
 


 


考马斯亮蓝染色
用去离子水短暂冲洗凝胶,用去离子水倾倒并加入足够的考马斯亮蓝染色剂以覆盖凝胶。在室温下轻轻摇动孵育凝胶至少1小时或过夜。将考马斯亮蓝染色剂倒入合适的容器中。污渍可以重复使用多次。                           
在去离子水中彻底冲洗凝胶,以去除残留的考马斯亮蓝染料,并用Destain 完全覆盖凝胶。在室温下轻轻摇动孵育凝胶,直到可见染色的蛋白带,并且背景很小,根据需要改变颜色。
 


数据分析


 


              该脱色凝胶可立即进行视觉和解释。在SDS-PAGE应用,磷酸化降低了流动性的Th é GST-酵母CTD 基板上。这是证明一个更高的表观分子量激酶处理样品相对于没有激酶控制样品。在半天然PAGE中,磷酸化增加了GST酵母CTD 底物的迁移率,这表现为在激酶处理的样品中表观分子量相对于无激酶的对照物的表观分子量较低。系统包括Chemi -Doc(Bio-Rad )和G:Box(Syngene )系统,可以在ImageJ中裁剪图像,也可以使用ImageJ在整个图像上均等地调整亮度和对比度,以提高凝胶的可解释性。 。应格外小心,以确保在所有考虑的样本上均明智且等效地应用图像修改。


                整篇文献均采用了EMSA分析方法,Mayfield 等人(2019)中介绍了该方法的实例,如图1C和图1C所示,将SDS-PAGE EMSA应用于两种GST-CTD底物。 2A。当需要提高分辨率并且要求对已建立的SDS-PAGE协议进行最小改动时,如补充图1D所示,半本地PAGE是一种有用的选择。


 


菜谱


 


LB中号(1公升)
将以下物质加入900毫升去离子水中:


              10克Bacto 胰蛋白tone


              5克酵母提取物


              10克氯化钠


              12克Bacto 琼脂(仅适用于琼脂平板)


摇动或搅拌溶液以溶解溶质
用去离子水将最终体积调节至1 L
在液体循环中于15 psi高压灭菌20分钟进行灭菌
如果需要,一旦培养基完全冷却(对于液体培养基)或已融化但摸起来舒适(对于琼脂平板),请添加抗生素,然后在无菌条件下将平板倒入
1,000x卡那霉素原液(50 mg / ml)
将500 mg卡那霉素单硫酸盐(MW 。= 582.60)溶于9 ml去离子水中
用去离子水将体积调节至10毫升
通过0.22过滤消毒溶液微米过滤器和分装成无菌试管             
储存在-20°C
1,000x IPTG库存(400 mM)
将950 mg异丙基-β-D- 硫代半乳糖苷(IPTG,MW 。= 238.30)溶于9 ml去离子水中
用去离子水将体积调节至10毫升
通过0.22过滤消毒溶液微米过滤器和分装成无菌试管
储存在-20°C
裂解缓冲液(1 升)
50 mM Tris-HCl pH 8.0(从1 M Tris-His pH 8.0储备液中稀释50 ml [ 配方14 ] )


              500 mM氯化钠(29.25 g)


              15 mM咪唑(从1 M库存中取15 ml )


              10%甘油(100%库存中有10 毫升)


              0.1%Triton X-100(从100%库存中取1 ml)


              的10mM β - 巯基乙醇(0.7 从14.3库存毫升M)


洗涤缓冲液(1 升)
50 mM Tris-HCl pH 8.0(从1 M Tris-His pH 8.0储备液中稀释50 ml [ 配方14 ] )


              500 mM氯化钠(29.25 g)


              15 mM咪唑(从1 M库存中取15 ml )


              的10mM β - 巯基乙醇(0.7 从14.3库存毫升M)


洗脱缓冲液(1 升)
50 mM Tris-HCl pH 8.0(从1 M Tris-His pH 8.0储备液中稀释50 ml [ 配方14 ] )


              500 mM氯化钠(29.25 g)


              400 mM咪唑(400万毫升,来自1 M库存)


              的10mM β - 巯基乙醇(0.7 从14.3库存毫升M)


透析缓冲液(1 L)
50 mM Tris-HCl pH 8.0(从1 M Tris-His pH 8.0储备液中稀释50 ml [ 配方14 ] )


              50毫米氯化钠(2.9 克)


              的10mM β - 巯基乙醇(0.7 从14.3股票毫升M)


对于FPLC应用中,缓冲器应通过一个0.4被过滤微米过滤器以除去任何颗粒。


4x激酶缓冲液(100 毫升)
              200 mM Tris-HCl pH 7.5(从1 M Tris-His pH 7.5储备液中稀释20 ml [ 配方14 ] )


              200 mM MgCl 2 (从2 M的储备液中提取10 ml )


4x ATP(5毫升)
22 mg腺苷5'三磷酸二钠盐水合物


              1 M Tris-HCl pH 7.5(可变)


将腺苷5'三磷酸二钠水合物溶于4 ml去离子水。
通过在pH 纸上点少量(1-2μl )溶液来验证pH 值。初始溶液应为酸性。
通过添加20-100调节pH用1M的Tris-HCl pH为7.5 微升每个addition.Once pH值寄存器7.5,调节溶液至5毫升最终体积之后的时间和检查pH使用去离子water.Adjusting pH值ATP解决方案至关重要。
4x GST- 酵母CTD 底物
              4 微克/ 微升GST-CTD酵母基质


              Mm Tris-50 HCl p H 8.0


4x P- TEFb 激酶溶液
              0.03 微克/ 微升CDK9 /周期蛋白T1蛋白,活性


              50 mM的Tris-HCl pH 7.5


4x TFIIH激酶溶液
              0.1 微克/ 微升CDK7 /细胞周期蛋白H / MAT1(CAK复合物)蛋白,活性


              50 mM的Tris-HCl pH 7.5


4x c- Abl 激酶溶液
              0.014 微克/ 微升C- Abl的激酶


              50 mM的Tris-HCl pH 7.5


1 M Tris-HCl pH 6.8(或7.5和8.0)(500 ml)
              60.6克Tris Base


              400毫升去离子水


结合Tris碱和去离子水


调节pH值至6.8 (或7.5和8.0)Ü 唱浓盐酸和使最终体积至500ml瓦特第i个额外的去离子水
室温保存
1.5 M Tris-HCl pH 8.8(500毫升)
              90.9克Tris Base


              400毫升去离子水             


结合Tris碱和去离子水
用浓盐酸将pH调节至8.8,并用去离子水使最终体积达到500 ml
室温保存
10%SDS(100毫升)
10克十二烷基硫酸钠(SDS)


              80毫升去离子水


将SDS和去离子水合并,搅拌直至溶解
用额外的去离子水将最终体积调节至100 ml
室温保存
2 倍Laemmli的样本缓冲区
              0.125 M Tris-HCl pH 6.8


              4%SDS(w / v)


              10%β - 巯基乙醇(V / V)


              20%甘油(v / v)


              0.02%溴酚蓝(w / v)


分离半天然PAGE / SDS -PAGE 凝胶(10%丙烯酰胺; 5 ml – 足够用于1凝胶,必要时可缩放)
              1.7毫升30%丙烯酰胺


              1.3毫升1.5M Tris-HCl pH 8.8


              1.9毫升去离子水


              50 微升10%SDS(W / V)(对于SDS-PAGE凝胶只)


              50 微升去离子水(为半天然PAGE凝胶上仅)


              50 微升10%过硫酸铵(重量/体积)


              4 微升TEMED


将30%的丙烯酰胺,1.5 M Tris-HCl pH 8.8,去离子水,SDS(用于SDS-PAGE)或其他去离子水(用于Semi-native PAGE)混合在螺口小瓶中,然后颠倒合并。
加入过硫酸铵和TEMED引发聚合反应,轻轻倒置,然后将溶液转移至准备好的Bio-Rad Mini-PROTEAN凝胶铸模中。
填充铸件,在凝胶顶部留出足够的空间用于堆叠层和梳子。
轻轻地在分辨凝胶的顶部涂上一层100%的乙醇,使其完全聚合并倒出乙醇。
堆叠式半天然PAGE / SD S-PAGE凝胶(5%丙烯酰胺,2 ml – 足以用于1凝胶,必要时可缩放)
              330 微升30%的丙烯酰胺


              250 微升1M的Tris-HCl pH 6.8的


              1.4毫升去离子水


              20 微升10%SDS(W / V)(对于SDS-PAGE凝胶只)


              20 微升去离子水(为半天然PAGE凝胶上仅)


              20 微升10%过硫酸铵(重量/体积)


              2 微升TEMED


将30%的丙烯酰胺,1 M Tris-HCl pH 6.8,去离子水和SDS(用于SDS-PAGE)或其他去离子水(用于Semi-native PAGE)合并在螺口小瓶中,然后颠倒合并。
加入过硫酸铵和TEMED引发聚合反应,轻轻倒置,然后将溶液转移至制备的含有聚合型分离凝胶的Bio-Rad Mini-PROTEAN凝胶铸件中。
用堆积的凝胶溶液填充铸件的其余部分,然后插入梳子。
让凝胶完全聚合。
将最终凝胶包裹在湿纸巾和保鲜膜中,在4 °C下保存2周。
1x Laemmli 运行缓冲液(Tris-甘氨酸)(1 L)
将以下物质加入900毫升去离子水中:


              3.03克Tris基础


              14.2克甘氨酸


              1毫升10%SDS(w / v)


让所有试剂溶解,必要时将pH调节至8.3。用额外的去离子水将体积调节至1 L.


1x天然Laemmli 运行缓冲液(Tris-甘氨酸)(1 L)
将以下物质加入900毫升去离子水中:


              3.03克Tris基础


              14.2克甘氨酸


让所有试剂溶解,必要时将pH调节至8.3。用额外的去离子水将体积调节至1 L.


考马斯亮蓝染色剂(100毫升)
结合以下内容:


              0.25克考马斯亮蓝R250


              45毫升乙醇


              45毫升去离子水


              10毫升冰醋酸


不断搅拌直至完全溶解


去污
结合以下内容:


              50毫升乙醇


              75毫升冰醋酸


              875毫升去离子水


搅拌或倒置容器,直到充分混合


 


致谢


 


这项工作得到了美国国立卫生研究院(Y01)的资助(R01 GM104896和125882)和韦尔奇基金会(Y-1Z的F-1778)的资助,此处介绍的方法是根据以前的工作改编的(Mayfield et al。,2019 )。


 


利益争夺


 


作者宣称没有任何竞争的经济利益。


 


参考文献


 


Corden,JL,Cadena,DL,Ahearn,JM,Jr.和Dahmus,ME(1985)。真核RNA聚合酶II最大亚基在羧基末端的独特结构.Proc Natl Acad Sci USA 82(23):7934 -7938。              
Dias,JD,Rito,T.,Torlai Triglia,E.,Kukalev,A.,Ferrai,C.,Chotalia,M.,Brookes,E.,Kimura,H. and Pombo,A.(2015)。的甲基化RNA聚合酶II非共识赖氨酸残基标志着哺乳动物细胞中的早期转录。生命4:11215。              
。尼莫,C.,巴塔耶,AR和Robert,F。(2013)作家,读取器,和RNA的功能聚合酶II的C-末端结构域的代码。化学启113(11):8491-8522。
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Copyright Mayfield et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mayfield, J. E., Irani, S. and Zhang, Y. (2020). Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates. Bio-protocol 10(12): e3648. DOI: 10.21769/BioProtoc.3648.
  2. Mayfield, J. E., Irani, S., Escobar, E. E., Zhang, Z., Burkholder, N. T., Robinson, M. R., Mehaffey, M. R., Sipe, S. N., Yang, W., Prescott, N. A., Kathuria, K. R., Liu, Z., Brodbelt, J. S. and Zhang, Y. (2019). Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb. Elife 8: 48725. 
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