Abstract
The endoribonuclease RNase E is believed to initiate the degradation of many mRNAs in Escherichia coli, yet the mechanism by which it recognizes cleavage sites is poorly understood. We have prepared derivatives of the mRNA encoding ribosomal protein S20 which contain a single major RNase E cleavage site at residues 300/301 preceded by variable 5' extensions. Three of these RNAs are cleaved in vitro with significantly reduced efficiencies relative to the intact S20 mRNA by both crude RNase E and pure Rne protein (endonuclease component of RNase E). In all three substrates as well as in the full-length mRNA the major cleavage site itself remains single-stranded. One such substrate (t84D) contains a 5' stem-loop structure characterized by three noncanonical A-G pairs. Removal or denaturation of the stem restores efficient cleavage at the major RNase E site. The other two contain single-stranded 5'-termini but apparently lack cleavage sites near the termini. Our data show that sensitivity to RNase E can be influenced by distant structural motifs in the RNA and also suggest a model in which the initial recognition and cleavage of a substrate near its 5' end facilitates sequential cleavages at more distal sites. The model implies that RNase E contains at least a dimer of the Rne subunit and that the products of the first cleavage are retained by Rne prior to the second cleavage.
Highlights
Ribonuclease (RNase) E has emerged as the principal endoribonuclease involved in the turnover of a number of mRNAs and some small RNAs
These data were interpreted to indicate that stem-loops can in some cases impede RNase E cleavage; similar conclusions have been drawn for derivatives of the mRNA encoding ribosomal protein S20 [10]
Effect of 5Ј Deletions on RNase E Cleavage of the mRNA for Ribosomal Protein S20 in Vitro—Previous work showed that sequences 5Ј to residue 178 in stem II of the synthetic S20 mRNA transcript t79D (Fig. 1) were dispensable for cleavage at residues 300/301/302 [4]
Summary
Templates and RNAs—Plasmid pGM87 containing the P2 leader, coding sequences, and rho-independent terminator of the gene for S20 in the vector pTZ18U [15] has been described previously (10; see Fig. 1). The forward primers, oligonucleotides 461 (5Ј-cgcagaattctaatacgactcactataggGCACAGAAAGCATTTAACGAAATG) or 444 (5Ј-cgcagaattctaatacgactcactataggGACAAAGCTGCTGCACAGAAAGCA), respectively, contain an EcoRI site and a T7 RNA polymerase promoter in addition to S20 sequences. The forward primer for pSM160, oligonucleotide 1110 (5Ј-ttacgaattcgatttaggtgacactataGAAATGCCAACCGGATCGTGG), contains an EcoRI site but an SP6 RNA polymerase promoter. Labeled RNA transcripts were purified by extraction with phenol/chloroform/ isoamyl alcohol (25:24:1) and two cycles of precipitation with ethanol in the presence of 2 M ammonium acetate. Samples containing unlabeled t84D and labeled RNA at 20 nM final concentration were renatured or boiled (treatments A and B above, respectively) and digested with one of the following enzymes in RNase E assay buffer [4] at 30 °C for times ranging from 3 to 10 min. Markers were prepared by heating labeled RNA for 5 min at 100 °C in 50 mM sodium carbonate, pH 9 (alkaline ladder), or by digesting labeled RNA supplemented with 3 g of carrier yeast RNA with 0.8 units of T1 RNase for 6.5 min at 50 °C in 50 mM sodium citrate, pH 5.0, containing 7 M urea (G ladder)
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