Cleavages in the 5′ region of the ompA and bla mRNA control stability: studies with an E. coli mutant altering mRNA stability and a novel endoribonuclease.

Abstract

We describe here the partial purification of a novel Escherichia coli endoribonuclease, RNase K. This protein catalyses site‐specific cleavages in the 5′ region of in vitro transcribed ompA and bla transcripts. Some of the resulting cleavage products are also found in cellular ompA mRNA, defining the in vivo activity of RNase K. The following evidence suggests that RNase K initiates mRNA degradation. First, RNase K cleavages are suppressed in the ams mutant, which has a generally prolonged mRNA half‐life. Secondly, RNase K cleavage products seem to have very short half‐lives in vivo, indicating that they are decay intermediates rather than processing products. Thirdly, the differences in in vivo half‐life between the ompA and bla mRNAs are mimicked in in vitro decay reactions with purified RNase K. The relationship between RNase K and the ams locus might point to a more general role of RNase K in mRNA degradation. We discuss the influence of mRNA secondary structure on RNase K cleavage specificity.

RNase E: still a wonderfully mysterious enzyme

“…While the particular RNA structural features that are recognized by the ARRBS are not known, it has been observed that interaction with the ARRBS can alter the higher-order structure of complex RNA substrates . The endonucleolytic activity of a 55 kDa me-dependent protein initially termed RNase K (Lundberg et a/., 1990) is now known to result from an RNase E breakdown product generated by proteolysis occurring either intracellularly or during protein isolation (Mudd and Higgins, 1993;Carpousis et a/., 1994;Lundberg et a/., 1995).…”

The ams-1 and rne-3071 temperature-sensitive mutations in the ams gene are in close proximity to each other and cause substitutions within a domain that resembles a product of the Escherichia coli mre locus

“…While there is no direct evidence that the ams gene encodes any ribonucleolytic activity, the temperature sensitivity of RNase E extracts isolated from the temperature-sensitive strains (6,16,17) and the increase in RNase E activity in cells containing additional copies of the ams gene (2) have led to the prevailing view that ams encodes RNase E (6) or a product required for RNase E activity (2). It has been argued that the ams dependency of some non-RNase E cleavages (11,15,23) in vivo is due to an aberrant action of the temperature-sensitive Ams protein at the nonpermissive temperature (6).Recently, the sequence of the entire ams gene has been published (4), extending and correcting previously reported sequences (7,9). Computer analysis has revealed limited homology between the C terminus of the Ams protein and several RNA-binding proteins, including a mitochondrial ribosomal protein from Neurospora crassa, the S1 ribosomal protein of E. coli, and the 70-kDa human small nuclear ribonucleoprotein (9).…”

“…While there is no direct evidence that the ams gene encodes any ribonucleolytic activity, the temperature sensitivity of RNase E extracts isolated from the temperature-sensitive strains (6,16,17) and the increase in RNase E activity in cells containing additional copies of the ams gene (2) have led to the prevailing view that ams encodes RNase E (6) or a product required for RNase E activity (2). It has been argued that the ams dependency of some non-RNase E cleavages (11,15,23) in vivo is due to an aberrant action of the temperature-sensitive Ams protein at the nonpermissive temperature (6).…”

Article

“…Cependant, les analyses par extension d'amorces ont permis d'identifier une région transcrite non traduite à l'extrémité 5' du gène clpX. Cette région non traduite pourrait avoir une influence sur la stabilité des ARNm et jouer un rôle sur la régulation post-transcriptionnelle [15]. En conséquence, les mécanismes de régulation des gènes de stress chez O. oeni semblent se distinguer de ceux établis chez Escherichia coli [1] et B. subtilis [5].…”