Effects of 3'-terminal phosphates in RNA produced by ribozyme cleavage.

In vitro runoff transcription using T7 RNA polymerase has been the method of choice to produce milligram quantities of RNA for structural studies+ Unfortunately, the T7 enzyme often adds one or more extra nucleotides at the 39 end, which results in a heterogeneous RNA product (Milligan et al+, 1987)+ This heterogeneity can be observed at the 59 end as well, depending on the transcription template (Ferre-D’Amare & Doudna, 1996)+ The lack of homogeneity, which potentially is deleterious for structural studies, can be overcome with the use of cis-and/or trans-acting ribozymes,which produce clean RNA ends after their catalytic reaction (FerreD’Amare & Doudna, 1996)+ This procedure has been scaled up for large quantities of RNA for NMR and X-ray crystallographic studies, and is useful for purification of larger RNAs (greater than 50 nt) where singlenucleotide resolution by gel electrophoresis is difficult+ Ribozymes that have been used in this manner include the hairpin ribozyme, the hammerhead ribozyme (HH), the hepatitis delta ribozyme (d), and the Neurospora varkud satellite RNA ribozyme (VS) (Guo & Collins, 1995; Price et al+, 1995; Ferre-D’Amare & Doudna, 1996)+All of these ribozymes leave a 59-hydroxyl and a 39-cyclic phosphate as products of cleavage+ During the course of a structural investigation of a selenocysteine insertion element from rat Type 1 iodothyronine 59-deiodinase (D1 SECIS), we wished to prepare milligram quantities of an RNA that comprised the upper stem/loop of this RNA structure (41 nt; Fig+ 1A)+ This stem/loop is a stimulator required for the insertion of selenocysteins at a specific internal UGA stop codon (Berry et al+, 1991)+ Construction of the upper stem/ loop RNA required that the base of the stem end with four noncanonical base pairs (Walczak et al+, 1996), with no additional base pairing below this terminal quartet, as determined from in vitro assays (Martin et al+, 1998)+ This RNA has been produced synthetically for crystallization trials, so we sought a thorough comparison between this molecule and the RNA produced by run-off transcription+ Because the RNA to be studied begins with an adenosine and ends in a uridine, a ribozyme was used at each end to produce homogeneous RNA+ The system used to accomplish this has been described previously (Ferre-D’Amare & Doudna, 1996) and the resulting T7-produced RNA would differ from the synthetic RNA only by the 39cyclic phosphate+ In making preliminary imino proton NMR assignments for both of these RNAs, we concluded that the base pairing for both RNAs is nearly identical, as evident from the one-dimensional NMR spectra collected in 90% H2O (Fig+ 2)+ Close inspection shows that there are subtle differences near the RNA helix end (bracketed region)+ The terminal U-U iminos of the ribozymecleaved RNA stem/loop are not observed at 25 8C, or these imino protons have altered chemical shifts and become degenerate with other iminos of the G-U base pairs (assignments to be presented elsewhere)+ In addition, the two preceding G-A base pairs change in chemical shift and are broadened, as evident in spectra collected at several temperatures (25 8C and 5 8C; Fig+ 2 and insets)+ The resonance broadening results in a loss of NMR detection at temperatures above 35 8C+ At the same temperature, these signals are detected with the synthetic RNA+ These NMR spectral differences were not due to either 39or 59-heterogeneities as determined by electrospray mass spectrometry of each RNA (Fig+ 2)+ The observed spectral differences can be interpreted in at least two manners+ The broadening could arise from flattening of the ribose pucker (O49-endo or O49-exo) imposed by the cyclic phosphate at the 39 terminus (as seen with cyclic nucleoReprint requests to: Ray Gesteland, University of Utah, Department of Human Genetics, 15 N 2030 E, Rm 6160, Salt Lake City, Utah 84112-5330, USA; e-mail: rayg@genetics+med+utah+edu+ RNA (1998), 4:607–612+ Cambridge University Press+ Printed in the USA+ Copyright © 1998 RNA Society+