Function in Escherichia coli of the non-catalytic part of RNase E: role in the degradation of ribosome-free mRNA.
RNase E contains a large non-catalytic region that binds RNA and the protein components of the Escherichia coli RNA degradosome. The rne gene was replaced with alleles encoding deletions in the non-catalytic part of RNase E. All the proteins are stable in vivo. RNase E activity was tested using a P(T7)-lacZ reporter gene, the message of which is particularly sensitive to degradation because translation is uncoupled from transcription. The non-catalytic region has positive and negative effectors of mRNA degradation. Disrupting RhlB and enolase binding resulted in hypoactivity, whereas disrupting PNPase binding resulted in hyperactivity. Expression of the mutant proteins in vivo anticorrelates with activity showing that autoregulation compensates for defective function. There is no simple correlation between RNA binding and activity in vivo. An allele (rne131), expressing the catalytic domain alone, was put under P(lac) control. In contrast to rne+,low expression of rne131 severely affects growth. Even with autoregulation, all the mutants are less fit when grown in competition with wild type. Although the catalytic domain of RNase E is sufficient for viability, our work demonstrates that elements in the non-catalytic part are necessary for normal activity in vivo.
Laboratoire de Microbiologie et Génétique Moléculaire (CNRS, UMR 5100), Université Paul Sabatier, 118 rue de Narbonne, 31062 Toulouse, France.
Anne
A
Leroy
Nathalie F
NF
Vanzo
Sandra
S
Sousa
Marc
M
Dreyfus
Agamemnon J
AJ
Carpousis
D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D017930:Genes, Reporter; D007763:Lac Operon; D020871:RNA Stability; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D017384:Sequence Deletion
D012329:RNA, Bacterial; D012333:RNA, Messenger; D004722:Endoribonucleases; C022537:ribonuclease E