Fig. 2.
Determination of the transcription start regions of the srpS and srpA genes in P. putida S12. (A) Overlap region of the srpS and srpA promoters. The translation start codons of srpS and srpA are indicated by arrows. The predicted transcription start sites for srpS and srpA (based on bioinformatics and RT-PCR analysis) are highlighted and designated +1PS and +1PA, respectively. The base pairs at the putative −10 and −35 positions of each promoter are bolded and underlined. The primers used in RT-PCR assays are underlined (or overlined for AF1), with names indicated in small font above or below the sequence. (B) RT-PCR results for determination of the transcription start region of srpS. Lanes 1 and 4, amplification with primer SF0 (expected size, 760 bp); lanes 2 and 5, amplification with primer SF1 (expected size, 694 bp); lanes 3 and 6, amplification with primer SF2 (expected size, 524 bp). (C) RT-PCR results for determination of the transcription start region of srpA. Lanes 1 and 4, amplification with primer AF2 (expected size, 346 bp); lanes 2 and 5, amplification with primer AF1 (expected size, 358 bp); lanes 3 and 6, amplification with primer AF0 (expected size, 371 bp). RNA templates in lanes 1, 2, and 3 of panels B and C were isolated from cells grown without toluene; RNA templates in lanes 4, 5, and 6 were isolated from cells grown with 20% (vol/vol) toluene.