Primer extension and Primer extension inhibition analysis (toeprinting)

The cnfY transcriptional start site was mapped by primer extension as described previously [33]. Primer cnfY_RO_rv was used for reverse transcription of RNA isolated from bacteria that were grown at 37°C. The DNA sequence reaction was performed with the same primer using the Thermo Sequenase cycle sequencing kit (USB, now Affymetrix) and plasmid pBO4465 as template.

Toe printing analysis was performed with 30S ribosomal subunit, in vitro transcribed RNA and tRNA^fMet (Sigma-Aldrich, St. Louis, MO, United States) according to a protocol described before [34]. A 5’-[³²P]-labeled cnfY-specific oligonucleotide cnfY_RO_rv, complementary to nucleotides +61 to +80 (from ATG) of the cnfY mRNA, was used as a primer for cDNA synthesis. The radiolabeled primer (0.16 ρmol) was annealed to the cnfY mRNA (0.08 ρmol) and incubated with 30S ribosomal subunit (24 ρmol) or Watanabe buffer (60 mM HEPES/KOH; 10.5 mM Mg(COO)₂; 690 mM NH₄COO; 12 mM β-mercaptoethanol; 10 mM spermidine; 0.25 mM spermine) in presence of tRNA^fMet (8 ρmol) at 25, 37 or 42°C for 10 min. After addition of 2 μl MMLV-Mix (VD+Mg²⁺ buffer, BSA, dNTPs and 800 U MMLV reverse transcriptase (Affymetrix, Santa Clara, CA, US), cDNA synthesis was performed for 10 min at 37°C. Reaction was stopped by addition of formamide loading dye and samples were separated on a 8% denaturing polyacrylamide gel. The Thermo Sequenase cycle sequencing Kit (USB, now Affymetrix) was used for sequencing reactions with plasmid pBO4465 as template and radiolabeled primer cnfY_RO_rv.