Ptz19r

The sequence of full-length mature 15S rRNA was amplified from yeast genomic DNA using primers 15S_F and 15S_R (all primer sequences are provided in the Supplemental Table S1), and Phusion DNA polymerase (Thermo Fisher), and cloned into pTZ19R (Thermo Fisher). The pTZ19::15S vector was linearized with SmaI (Thermo Fisher) and transcribed using the T7 transcription kit (Thermo Fisher), according to the manufacturer's instructions, with α-³²P-UTP (Hartmann Analytic) added to the reaction to produce radiolabeled substrate, followed by digestion with RNase-free DNase I (Roche). Templates for shorter RNAs were generated by annealing appropriate complementary synthetic DNA oligonucleotides (all sequences are provided in the Supplemental Table S1), containing the T7 promoter sequence, and transcribed in vitro as described above. The RNA transcripts were then purified by electrophoresis in denaturing acrylamide gels (5%–12%, depending on the transcript length) and isolation of the appropriate bands as described previously (Malecki et al. 2008 (link)).

gBlock gene fragments (IDT) containing biological sequence (100 nt upstream of, 50 nt downstream of the predicted serA and fisB terminator hairpins) flanked by two identical 27 nt C-less cassettes, inward-facing consensus promoters with extended −10 elements (P_forward and P_reverse), and EcoRI and HindII restriction digestion sites (NEB) were cloned into pTZ19R (Thermo Fisher). DNA templates containing both P_forward and P_reverse were PCR-amplified from the appropriate pTZ19R derivative using a primer pair that was specific for pTZ19R (M13_2.0 and M13_reverse_2.0, IDT, derivatives of M13 and M13 reverse universal sequencing primers). Conversely, DNA templates containing just P_forward were PCR-amplified from the appropriate pTZ19R derivative using a reverse primer specific for pTZ19R (M13_2.0) and a forward primer specific for the biological sequence (serA_uni/fisB_uni) (Figure 2—figure supplement 1). In vitro transcription termination reactions were conducted identically using both of the templates detailed in Figure 2—figure supplement 1.

A 290 bp fragment corresponding to the leader region of arpR, including the SD sequence, was amplified by PCR using primers ARRA-Up (5´ agt cgg atc cgg gta tcc ata tgg gga ag 3´) and ARRA-Lw (5´ gat caa gct tct ccg cct tgt acc aag aga 3´). The resulting PCR product was digested with HindIII and EcoRI and ligated into plasmid pTZ19R (ThermoScientific), previously digested with the same enzymes, to produce plasmid pARRA. Plasmids pARRA and pLM1 [9 (link)] were used as templates to amplify the DNA fragments containing arpR and sodB regulatory regions under the control of the T7 promoter. These PCR products were used to generate the arpR and sodB radioactively labeled transcripts in vitro using the TranscriptAid T7 Transcription Kit (ThermoScientific), following the manufacturer’s instructions, in the presence of [α-32P]CTP (PerkinElmer). Unlabeled RNAs were synthesized following the same protocol but with unlabelled CTP. RNA concentrations were estimated by UV absorption at 260 nm.