Structural requirements for the processing of Escherichia coli 5 S ribosomal RNA by RNase E in vitro

摘要: Abstract Processing of 9 S precursor RNA in Escherichia coli requires the endoribonuclease RNase E, which makes two cleavages to liberate p5, immature form 5 rRNA. The contributions primary and secondary structure E-mediated cleavage were investigated. 5′ domain was probed by partial ribonuclease digestion chemical modification. Our structural analysis supports a model spacer folds into tandem hairpins so that first processing site moiety resides stretch single-stranded residues. Site-directed mutagenesis cloned sequence performed synthetic transcripts derived from variety such mutant templates assayed as substrates for E-dependent endonuclease activity fractionated extracts. Partial or complete deletion did not eliminate site-specific RNA. Mutations affecting revealed upstream is important maintaining efficient processing. However, downstream either dispensable. results suggest E specifically recognizes cleaves sequences only when presented proper conformational context. Adjacent structures appear play direct critical role enzyme's recognition its substrate. Additionally, it may serve anchor regions ensure availability sites.