Academic journal article Genetics

Identification and Analysis of Escherichia Coli Ribonuclease E Dominant-Negative Mutants

Academic journal article Genetics

Identification and Analysis of Escherichia Coli Ribonuclease E Dominant-Negative Mutants

Article excerpt

ABSTRACT

The Escherichia coli (E. coli) ribonuclease E protein (RNase E) is implicated in the degradation and processing of a large fraction of RNAs in the cell. To understand RNase E function in greater detail, we developed an efficient selection method for identifying nonfunctional RNase E mutants. A subset of the mutants was found to display a dominant-negative phenotype, interfering with wild-type RNase E function. Unexpectedly, each of these mutants contained a large truncation within the carboxy terminus of RNase E. In contrast, no point mutants that conferred a dominant-negative phenotype were found. We show that a representative dominant-negative mutant can form mixed multimers with RNase E and propose a model to explain how these mutants can block wild-type RNase E function in vivo.

THE degradation of mRNAs in bacterial cells occurs rapidly, and mRNA half-lives of a few minutes or less are typical (COBURN and MACKIE 1999; STEEGE 2000). This instability has important consequences for gene expression and is believed to be important for cellular adaptation to changing environments. In E. coli, a number of potential ribonucleases that can participate in mRNA degradation and processing have been identified (DEUTSCHER and Li 2001; CONDON and PUTZER 2002). Among these factors, RNase E has been shown to have a major role in initiating the degradation of a large number of transcripts (COBURN and MACKIE 1999). In addition, RNase E plays important roles in the degradation of small regulatory RNAs and the maturation of transfer and ribosomal RNAs from their respective precursors (Li et al. 1999; Li and DEUTSCHER 2002; Ow and KUSHNER 2002; MASSE et al 2003).

RNase E is an endonuclease that cleaves mRNAs primarily within A-U rich, unstructured regions without the requirement for a stringent recognition sequence (LIN-CHAO et al. 1994; McDowALL et al. 1994). Its Nterminal domain contains a region of homology common to ribosomal Sl protein and to many other ribonucleases (Sl domain), as well as the amino acids necessary for RNA cleavage (McDoWALL and COHEN 1996; BYCROFT et al. 1997; SCHUBERT et al. 2004). A central region of the protein contains an arginine-rich region, similar to one found in many RNA-binding proteins (TARASEVICIENE et al. 1995). The C-terminal region participates in interactions with a number of other E. coli proteins including the exonuclease polynucleotide phosphorylase, RhIB helicase, and enolase, a glycolytic enzyme, to form a complex denoted the "degradosome" (CARPOUSIS etal. 1994; MICZAK et al. 1996; PY et al. 1996).

While the importance of RNase E in mRNA degradation is well appreciated, many details pertaining to RNase E function are still not well understood. For example, neither the specific amino acid residues that contribute to RNA cleavage nor those that discriminate substrates based on the RNA 5'-end have been identified. (LIN-CHAO and COHEN 1991; BOUVET and BELASCO 1992; MACKIE 1998). RNase E contains an arginine-rich, RNAbinding central domain, but its role in substrate recognition is unclear since RNase E variants lacking this domain remain capable of RNA cleavage (McDowALL and COHEN 1996). In addition, structural information on RNase E is presently limited to a 91 amino-acid region that encompasses the Sl domain (SCHUBERT et al. 2004). Therefore, it is not yet feasible to accurately model RNA cleavage by RNase E.

Some of these details could be clarified by studying defective RNase E variants. One recent study focused on the role of positively charged lysine and arginine as well as surface aromatic residues present within the Sl domain of RNase E (DiWA et al. 2002). Two other studies have investigated the effects of creating deletions within the C-terminal region of RNase E (Ow et al. 2000; LEROY et al. 2002). However, a systematic search for RNase E mutants remains to be undertaken. To provide a rapid means of identifying useful RNase E mutants, we have devised an efficient selection process, which is based on the ability of functionally defective but not functionally proficient RNase E variants to be overproduced without causing cell death. …

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