Overexpression of Cloned RhsA Sequences Perturbs the Cellular Translational Machinery in Escherichia coli

Abstract

ABSTRACT RhsA is a member of the multigene Rhs family and consists of a complex genetic sequence. This sequence consists of several distinct components, including a GC-rich core (core open reading frame [ORF]), an AT-rich extension (ext-a1) of the core ORF and an AT-rich region following the core extension (dsORF-a1). The functions of RhsA and the different distinct components, which can include open reading frames, are not well understood. Here, we study the effect of overexpression of the ext-a1 sequence and the ext-a1 3′ region, which includes a partial sequence of dsORF-a1, on Escherichia coli cells. Cells expressing these sequences show reduced cell growth and cell viability. The expression of these sequences dramatically affects different components of the transcription and translation machinery. Transcriptomic analysis reveals an increase in the expression of genes involved in transcription, RNA processing, and nucleotide biosynthesis and metabolism and a decrease in the expression of amino acid biosynthesis genes and transfer RNAs. Further, expression of the above-mentioned RhsA components increases ribosomal gene expression, as well as rRNA and ribosome abundance. Proteomic analysis reveals an overall reduction of protein expression at the genome-wide level in cells expressing the above-mentioned RhsA components. Based on these observations, we suspect a translation product of ext-a1 affects different regulatory mechanisms that control rRNA synthesis.