Movement protein of Apple chlorotic leaf spot virus is genetically unstable and negatively regulated by Ribonuclease E in E. coli

Scientific Reports
Rahul Mohan SinghVipin Hallan

Abstract

Movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) belongs to "30 K" superfamily of proteins and members of this family are known to show a wide array of functions. In the present study this gene was found to be genetically unstable in E. coli when transformed DH5α cells were grown at 28 °C and 37 °C. However, genetic instability was not encountered at 20 °C. Heterologous over expression failed despite the use of different transcriptional promoters and translational fusion constructs. Total cell lysate when subjected to western blotting using anti-ACLSV MP antibodies, showed degradation/cleavage of the expressed full-length protein. This degradation pointed at severe proteolysis or instability of the corresponding mRNA. Predicted secondary structure analysis of the transcript revealed a potential cleavage site for an endoribonuclease (RNase E) of E. coli. The negating effect of RNase E on transcript stability and expression was confirmed by northern blotting and quantitative RT-PCR of the RNA extracted from RNase E temperature sensitive mutant (strain N3431). The five fold accumulation of transcripts at non-permissive temperature (43 °C) suggests the direct role of RNase E in regulating the expression of ACLSV MP ...Continue Reading

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