Autogenous regulation of the Escherichia coli ksgA gene at the level of translation

Abstract

Various plasmids that contain the Escherichia coli ksgA gene, which encodes a 16S rRNA adenosine dimethyltransferase (methylase), were constructed. In one of these plasmids, the DNA encoding the N-terminal part of the methylase was fused to the lacZ gene, and in another construct, the ksgA gene contained a deletion which resulted in a truncated version of the methylase. When a cell contained one plasmid directing the synthesis of the intact, active methylase and another plasmid encoding the methylase-4-galactosidase protein, production of the latter product became strongly reduced. Likewise, synthesis of the truncated version of the methylase was diminished when the cell at the same time contained a plasmid producing the complete enzyme. These results were partly substantiated by in vitro experiments with a coupled transcription-translation assay system. By using a recently developed gel electrophoresis system for measuring protein-nucleic acid interactions, a specific binding of the ksgA methylase with its own mRNA could be established. Our results demonstrate that the expression of the ksgA gene can be, at least partly, autogenously controlled at the level of translation.

Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA

“…2A; see Experimental procedures ). The changes in growth in response to KsgA dosage is consistent with the location of ksgA in a highly regulated operon (van Gemen et al ., 1989; Roa et al ., 1989; Pease et al ., 2002), and implications of increased KsgA concentration and its potential impact in translation initiation are discussed below. To confirm that these data were due to KsgA and could not be attributed to the tagged version of the protein, overexpression experiments using both KsgA and E66A KsgA (see below) lacking epitope tags were performed.…”

Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA

“…2A; see Experimental procedures ). The changes in growth in response to KsgA dosage is consistent with the location of ksgA in a highly regulated operon (van Gemen et al ., 1989; Roa et al ., 1989; Pease et al ., 2002), and implications of increased KsgA concentration and its potential impact in translation initiation are discussed below. To confirm that these data were due to KsgA and could not be attributed to the tagged version of the protein, overexpression experiments using both KsgA and E66A KsgA (see below) lacking epitope tags were performed.…”

Dimethyl Adenosine Transferase (KsgA) Deficiency in Salmonella enterica Serovar Enteritidis Confers Susceptibility to High Osmolarity and Virulence Attenuation in Chickens

“…It has been reported that the ksgA gene has a weak promoter, lacks a recognizable ribosomal binding site (44), and displays an autogenous regulation during translation (45). Additionally, overexpression of ksgA in E.…”

Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase

“…Although KsgA does not methylate free 16S rRNA, it binds to a small fragment of rRNA containing the target adenosine bases (van Gemen et al, 1989). The RNA used in our study contains the 24 contiguous nucleotide residues of helix 45 (Figure 3A, middle panel) found in the 16S rRNA from E. coli .…”

Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA

“…2A; see Experimental procedures ). The changes in growth in response to KsgA dosage is consistent with the location of ksgA in a highly regulated operon (van Gemen et al ., 1989; Roa et al ., 1989; Pease et al ., 2002), and implications of increased KsgA concentration and its potential impact in translation initiation are discussed below. To confirm that these data were due to KsgA and could not be attributed to the tagged version of the protein, overexpression experiments using both KsgA and E66A KsgA (see below) lacking epitope tags were performed.…”

Dimethyl Adenosine Transferase (KsgA) Deficiency in Salmonella enterica Serovar Enteritidis Confers Susceptibility to High Osmolarity and Virulence Attenuation in Chickens

“…It has been reported that the ksgA gene has a weak promoter, lacks a recognizable ribosomal binding site (44), and displays an autogenous regulation during translation (45). Additionally, overexpression of ksgA in E.…”

Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase

“…Although KsgA does not methylate free 16S rRNA, it binds to a small fragment of rRNA containing the target adenosine bases (van Gemen et al, 1989). The RNA used in our study contains the 24 contiguous nucleotide residues of helix 45 (Figure 3A, middle panel) found in the 16S rRNA from E. coli .…”

Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA

“…2A; see Experimental procedures ). The changes in growth in response to KsgA dosage is consistent with the location of ksgA in a highly regulated operon (van Gemen et al ., 1989; Roa et al ., 1989; Pease et al ., 2002), and implications of increased KsgA concentration and its potential impact in translation initiation are discussed below. To confirm that these data were due to KsgA and could not be attributed to the tagged version of the protein, overexpression experiments using both KsgA and E66A KsgA (see below) lacking epitope tags were performed.…”

Dimethyl Adenosine Transferase (KsgA) Deficiency in Salmonella enterica Serovar Enteritidis Confers Susceptibility to High Osmolarity and Virulence Attenuation in Chickens

“…It has been reported that the ksgA gene has a weak promoter, lacks a recognizable ribosomal binding site (44), and displays an autogenous regulation during translation (45). Additionally, overexpression of ksgA in E.…”

Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase

“…Although KsgA does not methylate free 16S rRNA, it binds to a small fragment of rRNA containing the target adenosine bases (van Gemen et al, 1989). The RNA used in our study contains the 24 contiguous nucleotide residues of helix 45 (Figure 3A, middle panel) found in the 16S rRNA from E. coli .…”