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

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 DH5? 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 in E. coli.

SUBMITTER: Singh RM 

PROVIDER: S-EPMC5437062 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature