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Experiment 2.5. RNA Quantification, RNA Ladder by Nuclease Digestion, Lead Cleavage
(version: 03/14/00)

Background

The goal of this set of experiments is to verify that the product of your transcription reaction is tRNA. Before starting this lab you should be familiar with the 3D structure of tRNAs. In the first step of the lab you will quantitate your RNA by absorbance. You will then confirm the sequence with Rnase T1 digestion, to form a G ladder on a gel. Finally you will confirm the three dimensional structure with a cleavage reaction that is specific for properly folded tRNA. You will compare the structure of your tRNA transcript with that of in vivo produced tRNA, which contains extensive modifications.

RNA is a polyanion. Cationic metals interact strongly with RNA. Certain metals cause RNA degradation. Highly specific cleavage of tRNA molecules is observed in the presence of Pb and Eu. This activity is thought to contribute to metal toxicity in vivo. The reactions reflect specific features of RNA folding and provide an assay to probe three dimensional structure.

The Mechanism: A Pb (III) ion is thought to abstract a proton from a 2' OH group of a ribose, to form a 2' hydroxide anion. The 2' hydroxide anion is a strong nucleophile, that attacks the adjacent phosphorus atom. This attack forms an pentacoordinate intermediate, which has bipyramidal geometry. In this intermediate, the phosphorus atom is bonded to five oxygen atoms (O2', O3', O5', O1P and O2P). The intermediate decomposes to a 2' 3' cyclic phosphate, releasing a 5' hydroxide which is immediately protonated, to form a 5' hydroxyl. This step cleaves the phosphodiester backbone.

The specificity: Within a given fragment of RNA, Pb will cut some sites at much greater rates than others. The reaction requires extensive conformational rearrangement of the phosphodiester backbone. The phosphorous atom changes geometry in proceeding from starting material to intermediate to product. Therefore it is not surprising that the reaction rate of a given phosphodiester bond in an RNA fragment depends on conformational flexibility. Flexible loop regions a cleaved more readily than duplex regions. An additional factor cleavage efficiency is binding specificity. The reaction requires proper localization of the Pb ion on the RNA. Lead binds to the D loop of tRNA and cuts the T loop.

Outside Reading:
Biochemica et Biophysica Acta, 655 (1981), 89-95.  [Primary focus on intercalators, but deals with T1 digestion of tRNAphe].
Voet and Voet, 2nd edition, pages 967-971.

Word of Caution:
Contamination in the lab by "destructive" enzymes (proteases, DNAses, and RNAses) can cause problems with these and future experiments.  Care must be taken to avoid contamination.  Wear gloves when working with these enzymes.  Use a single pipette dedicated to RNAse T1.

Determination of RNA quantity

Acquire 2 microliters of RNA and dilute to 200 microliters.

Acquire an absorbance spectrum from 210 to 310 nm. Obtain the absorbance at 260 nm.

Determine the concentration of your RNA using A=ECL (remember that you diluted your sample 500-fold. Use the extinction coefficient of 5.0 x 10^5 M-1 cm-1 at 260 nM).  The molecular weight of tRNA is 21,000 grams/mole

Rnase T1 Digestion - Making a tRNA ladder

Materials

25 micrograms modified tRNA (refers to tRNA produced in vivo)
25 micrograms unmodified tRNA transcript (refers to tRNA produced by in vitro transcription)
100 mM Sodium Acetate, pH 5.5
100 mM Magnesium Acetate
100 mM Potassium Chloride
RNAse T1 (1 unit/microliter)

Procedure

Label four 600 microliter tubes (modified -, modified +, unmodified -, unmodified +)  The minus tubes will contain no RNAse and will be the control tubes.  The plus tubes will contain RNAse and will be the reaction tubes.

Add 12 microliters sodium acetate, 12 microliters magnesium acetate, 6 micoliters potassium chloride, and 40 microliters water to each tube.

Add 25 micrograms modified tRNA to the 'modified' tubes and 25 micrograms tRNA transcript to the 'unmodified' tubes.

PRE-prep tubes with 16 microliters 2X dye chilled on ice/water for each time point below for both the modified and modified reaction tubes.  PRE-prep one control tube for the modified and unmodified control tubes.

Time critical steps from here on!

Add 40 microliters RNAse T1 to the reaction tubes and 40 microliters water to the control tubes.

Immediately incubate all tubes at 37 degrees C.

Take 20 microliter samples at 1, 2, 4, 8, 16, 32, and 64 minutes from the reaction tubes.  Quench the reactions by placing in pre-prepped tubes with 16 microliters 2X dye chilled on ice/water.

Take a 10 microliter sample from the control tubes at the 64 minute stop time.

Gel (One for modified/One for unmodified)

Lane 1	control- No RNAse T1
Lane 2	1 minute digestion
Lane 3	2 minute digestion
Lane 4	4 minute digestion
Lane 5	8 minute digestion
Lane 6	16 minute digestion
Lane 7	32 minute digestion
Lane 8	64 minute digestion

Run a nucleic acid PAGE gel by the normal procedure, except load 20 microliters onto each lane and run the gel for ONLY 20 minutes at ~200 Volts.  Stain with Ethidium for 5 minutes.  Visualize and photograph the gel.

Lead Cleavage

Materials

25 micrograms modified tRNA and unmodified tRNA construct
100 mM HEPES, pH 7.45
100 mM Magnesium Chloride
100 mM Potassium Chloride
5 M Sodium Chloride
50 mM Lead Acetate

Procedure

Label four 600 microliter tubes (modified -, modified +, unmodified -, unmodified +)  The minus tubes will contain no lead and will be the control tubes.  The plus tubes will contain lead and will be the reaction tubes.

Add 8 microliters HEPES, 2 microliters Magnesium Chloride, 2 microliters Sodium Chloride to each tube.

Add 25 micrograms modified tRNA to the modified tubes and 25 micrograms tRNA transcript to the unmodified tubes.

Place all tubes on a float in a beaker containing 200 mL water at ~80 degrees.

Allow the beaker to cool on the benchtop to no warmer than 30 degrees C. (~30 minutes)

Prep tubes with 8 microliters 2X dye chilled on ice/water for each time point below for both the modified and modified reaction tubes.  Prep one tube for the modified and one for the unmodified control tube.

Time critical steps from here one!

Add 2 microliters of water to the control tubes and one microliter of water to the reaction tubes

Add 1 microliter of lead acetate to the reaction tubes.

Incubate at 25 degrees C, removing 10 microliter samples from each of the reaction tubes at 15, 30, and 60 minute intervals.

Take a 10 microliter sample out of the control tubes at the 60 minute stop time.

Quench the reactions IMMEDIATELY by placing in pre-prepped tubes of 8 microliter 2X loading dye in an ice bath. The loading dye solution contains urea and EDTA.

Gel Instructions

Lane 1	Yeast Phe mod- no lead
Lane 2	Yeast Phe mod- 15 minute cleavage
Lane 3	Yeast Phe mod- 30 minute cleavage
Lane 4	Yeast Phe mod- 60 minute cleavage
Lane 5	Yeast Phe unmod- no lead
Lane 6	Yeast Phe unmod- 15 minute cleavage
Lane 7	Yeast Phe unmod- 30 minute cleavage
Lane 8	Yeast Phe unmod- 60 minute cleavage

Run a "normal" nucleic acid PAGE gel for 30 minutes at ~250 Volts.  Stain with Ethidium for 5 minutes.  Visualize and photograph the gel.

Questions

tRNA Quantity Determination
1a.  Why is it important to purify your RNA transcript before quantifying it?  (Hint: What else in the transcription reaction would absorb at ~260 nm)
2a.  Why is it important to take a spectrum of your sample from 210 to 310 nm, instead of a single absorbance measurement at 260 nm.

RNAse T1 Digestion
1b.   Compare and contrast the digestion of the modified and unmodified tRNA.
2b.  Visualization of all cleavage products can be difficult.  Why is this? (Hint:  How does Ethidium bind to nucleic acids?)
3b.  We did not run the gel as long as normal.  Why?
 

Lead Cleavage
1b.  Compare and contrast the lead cleavage of the modified and unmodified tRNA.
2b.  According to the lead cleavage reaction, is you unmodified transcript folded properly?  If so, what does this say about the necessity of the modifications in the folding process?
3b.  We only saw the major cutting of the tRNA.  There are additional minor lead cutting sites.  The authors of these papers used autoradiography to see these fragments.  How does this work, i.e.. how does the tRNA become radioactive, how is it visualized, etc.?