Modulation of the activity of RNase E in vitro by RNA sequences and secondary structures 5' to cleavage sites.

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.

The role of RNA structure in determining RNase E-dependent cleavage sites in the mRNA for ribosomal protein S20 in vitro.

An RNA encompassing the 3' 147 residues of the mRNA for ribosomal protein S20 in Escherichia coli constitutes a naturally occurring degradative intermediate whose formation depends on RNase E. We have investigated the role of internal stem-loop structures in the RNase E-dependent cleavage which generates this product from S20 mRNA in a partially fractionated processing system in vitro. Individual stem-loops have been removed by deletion or destabilized by point mutations. No single hairpin structure is absolutely required for RNase E-dependent cleavage at the site 147 residues from the 3' end of the RNA. Primary sequences or secondary structures 5' or 3' to this site exert only a modest influence on the specificity of cleavage but can strongly modify its rate. Moreover, mutations in the S20 mRNA which destabilize stems 5' or 3' to the prominent cleavage site also reveal several strong cryptic RNase E cleavage sites. These data greatly strengthen the hypothesis that RNase E is a single-strand specific endoribonuclease. Our data further demonstrate that stem-loop structures adjacent to the prominent cleavage site are unlikely to provide a site of recognition for RNase E. Rather, they appear to stabilize (or "anchor") the local secondary structure so that the cleavage site is single-stranded and to occlude alternative sites so that the initial products of cleavage resist further attack.

RNase E activity is conferred by a single polypeptide: overexpression, purification, and properties of the ams/rne/hmp1 gene product.

Ribonuclease E, an enzyme that processes pre-5S rRNA from its precursor, is now believed to be the major endoribonuclease participating in mRNA turnover in Escherichia coli. The product of the ams/rne/hmp1 gene, which is required for RNase E activity, was overexpressed, purified to near homogeneity by electroelution from an SDS/polyacrylamide gel, and renatured. The purified polypeptide possesses nucleolytic activity in vitro with a specificity identical to that observed for crude RNase E preparations. In addition, both UV crosslinking and RNA-protein blotting unambiguously showed that the Ams/Rne/Hmp1 polypeptide has a high affinity for RNA. Our results demonstrate that RNase E activity is directly attributable to, and is an inherent property of, an RNA-binding protein, the ams/rne/hmp1 gene product.