Site-specific RNase E cleavage of oligonucleotides and inhibition by stem–loops

Abstract

The enzyme RNase E (ref. 1) cuts RNA at specific sites within single-stranded segments. The role of adjacent regions of secondary structure in such cleavages is controversial. Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I. These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites. Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access. Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites.

Promoter‐independent cold‐shock induction of cspA and its derepression at 37°C by mRNA stabilization

“…Note that in this region the eight-base sequence, AAUUAUUA, from bases 139-146, is in a single-stranded region in a bulge. This sequence may be an RNase E cleavage site as defined as an AU-rich region flanked by stable secondary structures (Naureckiene and Uhlin, 1996), although the requirement of secondary structures for RNase E cleavage is dependent upon its substrate structures (McDowall et al, 1995). It is important to note that a two-base substitution mutation from UAUUA to UUCUA by itself stabilized the mRNA at most by twofold, and, similarly, that the A to G mutation at base 147 also stabilized the mRNA only by 10-fold.…”

Determination of the Catalytic Parameters of the N-terminal Half of Escherichia coli Ribonuclease E and the Identification of Critical Functional Groups in RNA Substrates

“…As shown in Fig. 2, panel A, a 5-nucleotide 3Ј-(Fl)-labeled fragment produced by hydrolysis of p-BR10-Fl, a 10-nucleotide substrate corresponding in sequence to the RNaseE site at the 5Ј end of RNAI from pBR322 (29), can be separated readily. Moreover, this system can separate oligonucleotide substrates that differ only in the presence or absence of a monophosphate at the 5Ј end (data not shown).…”

“…In the first study of this enzyme that used oligonucleotide substrates, it was found that a decaribonucleotide (ACAGU2AUUUG) corresponding in sequence to the single-stranded region at the 5Ј end of RNAI, the antisense regulator of ColE1-type plasmid replication, is cut at least as efficiently as the corresponding full-length transcript by RNaseE (29). It was subsequently found for the 9 S precursor of 5 S rRNA that the "a" site (ACAGA2AUUUG) but not the "b" site (AUCAA2AUAAA) is cut efficiently (17).…”

A developmentally regulated Streptomyces endoribonuclease resembles ribonuclease E of Escherichia coli

“…The oligonucleotide BR13, containing the 13 nt single-strand sequence present at the 5Ј end of GGG.RNA I (McDowall et al, 1995), was labelled using T4 polynucleotide kinase (Life Technologies) (Sambrook et al, 1989) in the presence of [ 32 P0-␥]-ATP and was purified on urea-polyacrylamide gel (20%) (McDowall et al, 1995).…”

“…Cleavage assays for RNase E activity RNA cleavage assays were performed at 30ЊC in 50 l reaction mixtures, as described in McDowall et al (1995), except that 4 U of ribonuclease A inhibitor (RNasin; Promega) was also added. Reaction mixtures contained 10 fmol of the 32 Plabelled RNA substrate (synthesized as described above) and the indicated amounts of enzyme.…”