Mapping tRNA structure in solution using double-strand-specific ribonuclease V₁from cobra venom

Abstract

A method for mapping all base-paired stems in both elongation and initiator tRNAs is described using double-stranded-specific ribonuclease V1 from the venom of the cobra Naja naja oxiana. 32p-end-labeled RNA is first partially digested with double-strand-specific V1 nuclease under near physiological conditions, and the resultant fragments are than electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel run in 90% formamide. After autoradiography, the base-paired nucleotides are definitively located by comparing V1 generated bands with fragments of known length produced by both Neurospora endonuclease and base-specific ribonucleases. Using the substrates yeast tRNAPhe an E, coli tRNAfMet of known three-dimensional structure, we find V1 nuclease to cleave entirely within every base-paired stem. Our studies also reveal that nuclease V1 will digest paired nucleotides not hydrogen-bonded by standard Watson-Crick base-pairing. In yeast tRNAPhe cleavage of both wobble base-pairs and nucleotides involved in tertiary base-base hydrogen bonding is demonstrated.

Stem-loop structure in the 5′ region of potato virus X genome required for plus-strand RNA accumulation

“…Within SL1, four stem regions (SA, SB, SC and SD), three internal asymmetric loops (LA, LB and LC), and a terminal tetraloop (TL) are labeled. (Lockard & Kumar, 1981) was done at 25 C. Reaction conditions for both DMS modi®cation and RNase digestion experiments were optimized such that less than one nucleotide per RNA molecule was modi®ed. Products of these reactions were evaluated by primer extension with three oligonucleotide primers, eP1, eP2 and eP3, which are complementary to nt 78-97, 148-179 and nt 242-263, respectively. A summary of the data obtained by DMS modi®cation and RNase digestion of the 5 H 230 nt are superimposed on the most stable secondary structure prediction shown in Figure 1B.…”

Structural Features of MicroRNA (miRNA) Precursors and Their Relevance to miRNA Biogenesis and Small Interfering RNA/Short Hairpin RNA Design

“…These extra nucleotides when transcribed constitute the only differences between the proposed sequences of natural pre-miRNAs (3) and these investigated in this study. (27), and Pb 2ϩ ions (28 -30) and nucleases included the singlestrand-specific S1, T1, and T2 (31) and double-strand-specific V1 (32,33). The metal ions differentiate between rigid and flexible regions of the RNA structure.…”

Regulation of a double-stranded RNA modification activity in human cells.

“…When 32P-labeled poly(I-C) was used in the cell treatments, only 6 ng (0.3 ,ug/ml) of residual 32P-labeled poly(I C) remained in 10 pAl of extract, more than 100-fold less than the amount necessary to cause the levels of inhibition observed (see Materials and Methods). In addition, inactive extracts were preincubated with cobra venom ribonuclease (25) to degrade any preexisting dsRNA, and the ribonuclease was inactivated before the assay was performed. Such treatment did not restore dsRNA modification to inactive extracts.…”