PMID:11952897
| Citation |
Lee, K, Bernstein, JA and Cohen, SN (2002) RNase G complementation of rne null mutation identifies functional interrelationships with RNase E in Escherichia coli. Mol. Microbiol. 43:1445-56 |
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| Abstract |
The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have sequence similarity and broadly similar sequence specificity. Whereas the absence of Rne normally is lethal, we show here that E. coli bacteria that lack the rne gene can be made viable by overexpression of Rng. Rng-complemented cells accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase P (i.e. M1 RNA), indicating that normal processing of these Rne-cleaved RNAs was not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from precursors by RNase G cleavage in vitro. Using DNA microarrays containing 4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose steady-state level was affected by Rne, Rng or the N-terminal catalytic domain of RNase E. Most transcript species affected by RNase E deficiency were also elevated in an rne deletion mutant complemented by Rng. However, approximately 100 mRNAs that accumulated in Rne-deficient cells were decreased by rng-complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality. Remarkably prominent in this group were mRNAs implicated in energy-generating pathways or in the synthesis or degradation of macromolecules. |
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| Keywords |
Base Sequence; Endoribonucleases/genetics; Endoribonucleases/metabolism; Escherichia coli/genetics; Escherichia coli/growth & development; Escherichia coli/metabolism; Escherichia coli Proteins; Gene Deletion; Genetic Complementation Test; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; RNA, Bacterial/genetics; RNA, Bacterial/metabolism; RNA, Catalytic/metabolism; RNA, Messenger/metabolism |
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Useful Materials and Methods
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Strains and plasmids used
The isogenic E. coli K-12 strains used are derivatives of N3433 (lacZ43, relA, spoT, thi-1) (Goldblum and Apirion, 1981).
KW153 (Wang and Cohen, 1994) = N3433 (lacZ43 relA spoT thi-1) rne::cat
KSL1000 = N3433 (lacZ43 relA spoT thi-1) rne::cat recA::Tn10
KSL1004 = N3433 (lacZ43 relA spoT thi-1) rne::cat recA::Tn10 rng(del)::FRT-Kan-FRT
pBAD-RNE is a derivative of pSC101 (Cohen andChang, 1973; 1977) that directs the conditional synthesis of a full-length carboxy-terminally tagged form of E. coli RNase E under control of the BAD promoter.
pLAC-RNE2 is a derivative of pPM30 (Meacock and Cohen, 1980) that contains the same RNase E gene of pBAD-RNE under control of lacUV5 promoter.
pNRNE5 is a derivative of pLAC-RNE2 which encodes a carboxyterminally tagged, truncated form of RNase E lacking the C-terminal 562 residues (N-RNase E).
pRNG3 encodes E. coli RNase G with a hexahistidine affinity tag at the C-terminus; changes in RBS.
Annotations
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