Experiment Summary

This protocol describes the coupling of (i) "live" in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box leader RNA, a transcription control system, folds into a thermodynamically very stable stem-loop structure without the tRNA present, which makes in vitro binding interaction of both pre-formed RNAs very difficult. I therefore adjusted the binding assay to mimic the "natural" situation in the bacterial cell, where the pre-formed, stable tRNA is already present while the T-box leader RNA is actively transcribed by the RNA polymerase. The first part of the protocol also describes the in vitro transcription and labeling of the tRNA.

Materials and Reagents

A. For in vitro transcription (IVT) in general

1. T7 RNA polymerase including 5x T7 transcription buffer
2. RNase inhibitor
3. 100 mM of ATP, UTP, GTP, CTP (NTP set)
4. Recombinant DNase I
5. RNase-free water
6. [alpha 32P]-CTP (800 Ci/mmol, 10 µCi/µl)
7. 1x T7 transcription buffer

B. For tRNA IVT in particular

1. PCR Purification Kit
2. Guanosine 5'-monophosphate disodium salt hydrate from yeast (GMP) (as 100 mM stock solution in RNase-free water)
3. Bio-Spin chromatography columns, Bio-Gel P-6 in Tris buffer
4. OptiPhase Supermix liquid scintillation cocktail
5. Microbeta Starter kit plates

C. For denaturing Urea-PAGE

1. Urea
2. Rotiphorese 40% acrylamide/bis-acrylamide (19:1) solution
3. Ammonium persulfate (APS)
4. N, N, N', N'-Tetramethylethylenediamine (TEMED)
5. 2x MOPS buffer
6. Formamide
7. Formaldehyde
8. Saccharose
9. Bromophenol blue
10. Xylencyanol
11. 1x TBE buffer
12. 2x RNA loading dye

D. For native polyacrylamide gel electrophoresis (PAGE)

1. 40% acrylamide/bis-acrylamide (19:1) solution, APS and TEMED as listed in C
2. Glycerol
3. Tris
4. Boric acid
5. EDTA
6. 0.5x TBE buffer
7. 10x loading buffer

Equipment

A. Basic lab equipment for molecular biology with emphasis on RNase-free

1. 0.5 ml RNase-free microfuge tubes
2. 1.5 ml RNase-free microfuge tubes
3. Microliter pipetteswith tips and 15 ml or 50 ml tubes
4. Table-top centrifuge
5. Heating block for 1.5 ml and 0.5 ml tubes

B. Lab equipment in designated area for radioactive work

1. Liquid scintillation counter
2. Electrophoresis chamber, glass slides, spacers, clamps and combs for vertical PAGE
3. Plastic wrap, transparent sheets and Whatman paper (e.g. Sigma-Aldrich, catalog number: Z742422 )
4. Gel dryer (Model 583 Gel Dryer) with vacuum pump
5. PhosphorImager and PhosphorImager screen

Procedure

A. In vitro transcription (IVT) of tRNAs with free 3'cca end

1. Primer for amplification of the tRNA template from the bacterial chromosome by PCR need the T7 promoter sequence at their 5' end for in vitro transcription by the T7 polymerase. For successful transcription, three purine bases, at best guanine, should follow the core promoter sequence (underlined): nnnnCTAATACGACTCACTATAGRRnnnnnn.… with the first G (in bold) as transcription start.

2. PCR products for tRNA templates are cleaned up with the MinElute PCR Purification Kit according to the manufacturer's protocol.

3. The tRNA IVT reaction is set up in 20 µl volume as follows.

a) Add 1x T7 reaction buffer, 20 U RiboLock™ RNase inhibitor, 0.5 mM ATP, GTP and UTP, 12 µM CTP, 9 mM GMP, 20 U T7 RNA polymerase and 2 µl of [alpha 32P]-CTP (800 Ci/mmol, 10 µCi/µl) to 4 pmol of DNA template (tRNA-PCR).

b) Incubation of the reaction mix at 37 °C for<7 h to enrich transcription of short fragments and stop of reaction at -20 °C for 5 min.

c) DNA template is removed by adding 1 U of recombinant DNase I and incubating at 37 °C for further 30 min.

d) tRNA products have to be cleaned up using e.g. the Micro Bio-Spin™ chromatography columns with Bio-Gel P-6 in Tris buffer to ensure no small tRNA fragments are lost. Keep 1 µl of the reaction volume before the clean-up if you use the scintillation counter to measure the incorporation rate.

4. To establish the incorporation rate of radionucleotides during IVT into the produced tRNA, 1 µl of the reaction volume before and after clean-up is used for scintillation count.

a) OptiPhase Supermix liquid scintillation cocktail (50 µl per well) are pipetted into a rigid 96-well plate and the 1 µl samples are added, with one empty well between samples to avoid cross-counting of wells.

b) In parallel, a dilution series of the utilized radionucleotide [alpha 32P]-CTP is added to the same plate to obtain a standard curve for the cpm (counts per min).

c) The plate is sealed and placed in the liquid scintillation counter. The cpm from the standard curve will give the cpm value per µCi. This will change with each experiment, due to the half life of P32. The cpm values of the sample before and after clean-up helps to calculate the incorporation rate of radionucleotides and subsequently the RNA yield after IVT to determine the molarity of tRNA to be used in the binding assay.

B. Recipe for denaturing Urea-PAGE

1. A 10% (w/v) acrylamide/ 8 M urea gel was chosen for the separation of 78 nt tRNA molecules on a small size gel (10 x 10 cm).

a) 3.36 g urea are solved in 1x TBE (up to volume of 5.25 ml) by stirring for > 4 h at room temperature. 1.75 ml 40% acrylamide/bis-acrylamide (19: 1) solution are added to the 8 M urea solution to reach 7 ml total volume.

b) Clean glass slides of desired size are set up with spacer on left and right and either put in a gel casting chamber or fixed with clamps on both sides.

c) 35 μl [0.5% (v/v)] of 10% APS and 15 μl 0.2% (v/v) TEMED are added to the urea-acrylamide mix to catalyze the polymerization process.

d) The mix is quickly poured between the glass slides (e.g. with 10 ml glass pipette) and the comb with desired amount of wells adjusted in the gel.

2. The fully polymerized gel was pre-run in 1x TBE buffer at 250 V at room temperature for 40 min. Remove the comb for that.

3. tRNA samples were mixed with 2x RNA loading dye and heat denatured at 85 °C for 5 min before loading.

4. Electrophoresis settings are 250 V at room temperature to run for 1 h in 1x TBE buffer.

5. After electrophoresis run the gel is transferred to Whatman paper and covered in a transparent sheet before exposure of a PhosphorImager screen (couple of hours is usually sufficient), followed by scan with the PhosphorImager FLA-7000 (see Figure 1).

C. IVT-RNA-tRNA binding assay

1. A typical 10 μl reaction volume contains all components for T7 in vitro transcription:

a) 1x T7 transcription buffer, 10 U of RiboLock RNase inhibitor, 6 U of T7 RNA polymerase and 0.5 mM NTPs (ATP, UTP, GTP, CTP). DNA template (PCR product) for the leader RNA is added to 8 nM and the radiolabeled tRNA to 50 nM.

b) Control reactions are set up accordingly without tRNA but with 12 µM CTP and 1 µl [alpha 32P]-CTP (800 Ci/mmol, 10 µCi /µl) to check for IVT efficiency.

c) The reaction mix is incubated at 37 °C for 2 h, which is sufficient for the 440 nt transcript.

d) 2 µl of 10x loading buffer are added and the samples immediately loaded onto a non-denaturing polyacrylamide gel.

2. A 6% (w/v) acrylamide gel was chosen to separate a 78 nt tRNA from the complex with a ~440 nt RNA on a medium size vertical gel.

a) Clean glass slides of desired size are set up with spacer on left and right and either put in a gel casting chamber or fixed with clamps on both sides.

b) 1.5 ml 40% acrylamide/bis-acrylamide (19:1) solution and 0.5 ml 10x TBE are mixed with RNase-free water to 10 ml volume and then 40 μl [0.4% (v/v)] of 10% APS and 20 μl [0.2% (v/v)] TEMED are added to catalyze the polymerization process.

c) The mix is quickly poured between the glass slides (e.g. with 10 ml glass pipette) and the comb with desired amount of wells adjusted in the gel. Avoid air bubbles!

d) The fully polymerized gel has to be pre-run in 0.5x TBE buffer at 200 V at 4 °C for at least 40 min. Remove the comb for that. Rinse the wells before loading samples.

e) Immediately load the samples after binding reaction.

f) Electrophoresis settings are 100 V (= 6.7 V/cm), 5 W at 4 °C to run for >3 h.

3. After electrophoresis, the gel is transferred onto a Whatman paper and covered with plastic wrap, then dried by vacuum gel dryer at 80 °C for < 30 min.

4. Detection of the radiolabeled RNA complex is achieved by exposure of a PhosphorImager screen (often over night), followed by scan with the PhosphorImager FLA-7000 (see Figure 2).

Representative Data

Figure 1. Scanned image of denaturing Urea-PAGE of labelled tRNAs.

Figure 2. Scanned image of native PAGE gel after binding assay.