Figure 1. GalE is tagged with the SsrA peptide at several sites.

A) Schematic representation of the galE transcripts used in this study. GalE was produced from constructs with and without downstream galT́ coding sequence. His₆-GalE variants were also expressed from related constructs encoding an N-terminal hexa-histidine (his₆) epitope tag. The intrinsic transcription terminator from the E. coli trp attenuator (trp-At) was introduced downstream of galE as described in the Methods. Northern blot probe binding sites and the positions of codon-192 and codon-338 truncations in the in vitro transcript standards are indicated. B) Western blot analysis of SsrA(DD) tagging. Whole-cell lysates from induced (+IPTG) and uninduced (-IPTG) cells were analyzed by Western blot using anti-SsrA(DD) polyclonal antibodies. The His₆-GalE samples were purified by Ni²⁺-NTA affinity chromatography prior to analysis. Full-length GalE tagged at the C-terminus is indicated as GalE-SsrA(DD). C) Mass spectrometry of SsrA(His₆)-tagged GalE. SsrA(His₆)-tagged GalE chains were purified as described in Methods and analyzed by electrospray ionization-mass spectrometry. SsrA(His₆) tagging occurs at positions corresponding to GalE residues Val127 – Leu154. The identified tagging clusters are presented schematically on the GalE chain. D) Northern blot analysis of galET́ and galE(trp-At) mRNA. Total RNA was isolated from tmRNA⁺ and ΔtmRNA cells and analyzed by Northern blot as described in Methods. The in vitro transcripts lane contains a mixture of two galE transcripts that are truncated at codon-192 and codon-338 of galE.