FIG 1.

SecA copurifies with mRNAs encoding nascent Sec substrates. (A) Cells expressing His-SUMO-SecA796* and an orthologous tRNA-tRNA synthetase system evolved to recognize Bpa were incubated in the presence (+) or absence (−) of UV light at 365 nm. Treatment with UV light resulted in the appearance of at least one high-molecular-mass adduct (*), which was purified using an Ni affinity column. The running positions of full-length His-SUMO-SecA (Full-length) and the truncated peptide in which translation terminated at codon 796 (Truncated) are indicated. (B) RT-PCR analysis of UV-treated, StrepTactin-purified Strep-SUMO-SecA796* using primers specific for the messages encoding Braun's lipoprotein (lpp), PhoE, OmpF, TolC, OmpC, and thioredoxin-1 (trxA). Top, reaction mixtures containing complete RT-PCR mixture (including reverse transcriptase). Bottom, PCR mixtures in which reverse transcriptase was omitted. (C) Histogram of enrichment scores for mRNAs that copurify with SecA. Strep-SUMO-SecA796* was cross-linked to ribosomes as in the experiment whose results are shown in panel A, ribosomes were purified from cell lysates by ultracentrifugation (total ribosomes), and the Strep-SUMO-SecA796*-cross-linked ribosomes were purified using a StrepTactin column (SecA-cross-linked ribosomes). The mRNAs from the total ribosomes and SecA-cross-linked ribosomes were isolated by RT-PCR using a poly(A) primer and hybridized to an E. coli microarray. The enrichment score is the log₂ of the signal from the purified SecA-cross-linked fraction divided by the signal from total ribosome samples. Gray bars, all messages; black bars, messages for proteins in gene ontology category GO:0005886 (plasma membrane).