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.The Rhs family in Escherichia coli consists of five genetic elements. These elements are found in many, but not all, natural E. coli strains (4, 7, 11, 16). The Rhs (or rearrangement hot spot) elements were originally detected as components of recAdependent intrachromosomal recombination (8). Subsequent studies have defined the Rhs loci on the chromosome and elucidated the evolutionary history of these elements (4,6,7,19). Members of the Rhs family share one or more discrete homologous structures, one of which, a 3.7-kb GC-rich core, is common to all the elements. The Rhs family also contains unique sequences specific to each element, such as dissimilar sequences that flank the core of Rhs elements. Certain components of the Rhs elements also have a distinct GϩC content (approximately 40% or 60%) compared to the rest of the E. coli genome (approximately 50%), suggesting their origin is outside the E. coli species (4). While the function of Rhs elements is not fully understood, the amino acid sequence suggests that the elements encode hydrophilic proteins with repetitive sequence elements and divergent C termini (7).RhsA, a member of the Rhs family, is 8.2 kb long and consists of some distinct sequence features. The RhsA sequence can include five different open reading frames (ORFs). The 3.7-kb GC-rich core includes a long core ORF that extends 417 bases into a 1.3-kb AT-rich region. This extension is designated exta1. The 1.3-kb AT-rich region also contains dsORF-a1, an 840-bp ORF. Both ext-a1 and dsORF-a1 contain a high number of infrequently or rarely used codons; ...