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J Mol Microbiol Biotechnol. 2012;22(3):135-46. doi: 10.1159/000339832. Epub 2012 Jul 24.

Identification of two DNA helicases UvrD and DinG as suppressors for lethality caused by mutant cspA mRNAs.

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  • 1Department of Biochemistry, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.

Abstract

CspA is a major cold shock-inducible protein (70 aa), and its major role in the cold shock response was shown to be as an RNA chaperone destabilizing secondary structure of mRNAs at low temperature. Previously, we showed that the overexpression of mutant cspA containing premature non-sense codons at various positions led to stalled ribosomes on mutant cspA transcripts, ultimately leading to cell death. This lethality is primarily due to the highly translatable cspA 5'-UTR that recruits most of the ribosomes from other mRNAs, which are then stalled at the abnormal stop codon. This was called the 'LACE' effect. We show here that non-sense mutation even at the 67th position as well as substitutions of aromatic amino acid residues present on the RNA-binding surface of CspA protein to alanine caused the LACE effect by trapping a substantial amount of ribosomes on cspA mRNAs. In an attempt to identify a suppressor(s), which may help the cells to recover from the inhibitory LACE effect, genetic screening of an Escherichia coli genomic library was performed. We isolated suppressors that contained the genomic fragments encoding uvrD and dinG, respectively, whose gene products are ATP-dependent DNA helicases. The nucleic acid-binding and ATPase activities of these two helicases were found to be essential for their suppression activity. This genomic screening offers an approach to shed light on the mechanistic of 5'-UTR of cspA mRNA and novel roles of E. coli helicases that function in DNA repair.

Copyright © 2012 S. Karger AG, Basel.

PMID:
22832783
[PubMed - indexed for MEDLINE]
PMCID:
PMC3480728
Free PMC Article
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