FIG. 4.
Relevant DNA sequences for transcription and translation of the as-48ABC operon and determination of the initiation of transcription and mRNA processing. (A) The PA promoter of the as-48ABC operon and RBSs, as well as the initiation and termination translational codons, are underlined. The long IR is marked by arrows. The three bases changed to generate the XhoI site are in the gray box. Transcription initiation sites of as-48A (inferred from results presented in panel B) are underlined and labeled as +1. The processing sites in the mRNA (inferred from results presented in panel C and corresponding to positions 401 and 402 of the mRNA) are indicated by arrowheads. Also indicated are the location, sequence, and polarity of all primers used for RT-PCR experiments depicted in Fig. 2 (F-AsAB and R-AsAB) and 3 (F-AsA, F-AsAB, F-AsBC, R-AsA, R-AsAB, and R-AsBC) and for primer extensions depicted in panel B (R-5′ mRNA) or in panel C (R-processing). F, forward; R, reverse. Determination of the initiation of transcription from PA (B) and mRNA processing (C) by primer extension in the wild-type and pAM401-81X mutant strains. Autoradiograms of the gels are depicted, and the detected extended products are labeled with the corresponding coordinates in the as-48ABC mRNA. The coordinates were inferred from the length of the products (95 nt for +1 in panel B and 70 and 69 nt for 400 and 401 in panel C) that were estimated by the use of unrelated DNA sequence (A, C, T, and G) ladders.