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Full-length S1 was PCR-amplified from Escherichia coli X90 genomic DNA by using upstream (5’-TAT ACA TAT GAC TGA ATC TTT TGC TCA ACT C-3’) and downstream (5’-ATA TCT CGA GCT CGC CTT TAG CTG CTT TGA AAG-3’) primers (NdeI and XhoI sites in bold). DNA for each S1 fragment was PCR-amplified from the plasmid encoding the full-length protein. A methionine codon was added to the 5’-end of the R2–R4, R3–R4, R2–R3, R2, R3, and R4 coding sequences. The N, N–R1, N–R2, and N–R3 variants were PCR-amplified with the S1 upstream primer and downstream primers 5’-GCA TTC TCG AGT TCC ATG CCT TCC TGG TTT TCC-3’ (N, note that this primer results in deletion of residues E181 and R182 and insertion of an Asp residue at this position), 5’-GCA TTC TCG AGG TCG GTC AGG TTG GTC ACG-3’ (N–R1), 5’-GCA TTC TCG AGA CGG TCG CCC TTG TTG TGG G-3’ (N–R2), and 5’-GCA TTC TCG AGG ATA CGG CCT TTC TTG TTC AGA GC-3’ (N–R3). The R1–R4, R2–R4, R3–R4, and R4 variants were PCR-amplified with the S1 downstream primer and upstream primers 5’-GCA TTC ATA TGG AAG TTA AAG GTA TCG TTA AGA ACC-3’ (R1–R4), 5’-GCA TTC ATA TGG TAG CTA TCG CTA AAC GTT ATC CGG-3 (R2–R4), 5’-GCA TTC ATA TGC AGC AGT TCG CGG AAA CCC AC-3’ (R3–R4), and 5’-GCA TTC ATA TGG CAG AAG ATC CGT TCA ACA ACT GG-3’ (R4). The R1, R1–R2, and R1–R3 variants were PCR-amplified with the R1–R4 upstream primer and the N–R1, N–R2, and N–R3 downstream primers, respectively. The R2–R3 and R2 variants were PCR-amplified with the R2–R4 upstream primer and the N–R3 and N–R2 downstream primers, respectively. The R3 variant was PCR-amplified with the R3–R4 upstream primer and the N–R3 downstream primer. Coding sequences for ribosomal protein S1 and truncated variants were cloned under control of the T7 RNA polymerase promoter between the NdeI and XhoI sites of pET21b (Novagen), resulting in a His₆ tag at the C terminus of each protein.

pCH405-FLAG-AraC was generated in a manner similar to pCH410 (1) from pCH206. Plasmid derivatives of pCH410 (1) encoding S1, N, N–R1, R1–R4, and R2–R4 under the control of the pBAD promoter were generated by digestion of the protein coding sequences from pET21b with StyI, end-filling with Klenow DNA polymerase, and digestion with NdeI followed by ligation with NdeI- and HpaI-digested pCH410 (1). Plasmid derivatives of pCH405-FLAG-AraC encoding S1, N, N–R1, N–R2, R1–R4, and R2–R4 were generated in a similar manner by ligation into pCH405-FLAG-AraC.

pPW500-leaderless was constructed by PCR amplification of pPW500 with 5’-phosphorylated upstream and downstream primers 5’-ATG GGC ACA AAA AAG AAA CCA TTA ACA C-3’ and 5’-AAT TCC ACA CAT TAT ACG AGC CGG-3’, respectively. pPW500-extended-SD was constructed by PCR amplification of pPW500 with 5’-phosphorylated upstream and downstream primers 5’-CAG ACC ATG GGC ACA AAA AAG AAA CC-3’ and 5’-TCA CCT CCT TGT GTG AAA TTG TTA TCC GCT CAC AAT TCC-3’, respectively. PCR products were digested with DpnI to remove the template plasmid, purified on agarose gels, and ligated with T4 DNA ligase.

The tmRNA transcription template was amplified by PCR from plasmid pKW11 (2) with upstream and downstream primers 5’-GAA TTC TAA TAC GAC TCA CTA TAG GGG CTG AT-3’ (T7 promoter in bold) and 5’-TGG TGG AGC TGG CGG GAG TTG AAC-3’, respectively. The arc-st11 mRNA transcription template was PCR-amplified from plasmid pET800 (3) with primers 5’-GAA ATT AAT ACG ACT CAC TAT A-3’ and 5’-GCT AGT TAT TGC TCA GCG GTG GC-3’.

1. Hayes, C. S. & Sauer, R. T. (2003) Mol. Cell 12, 903–911.

2. Keiler, K. C., Waller, P. R. H. & Sauer, R. T. (1996) Science 271, 990–993.

3. Milla, M. E., Brown, B. M. & Sauer, R. T. (1994) Struct. Biol. 1, 518–523.