Fig 2.

3′-end and 5′-end S1 nuclease mapping of the ureB 5′ region. (A) Physical map of the ureAB gene cluster with an antisense sRNA complementary to the 5′ one-third of ureB. The corresponding transcripts for the ureAB genes (intact 2.7 kb and truncated 1.4 kb) are shown at the top. The 3′-end-labeled and 5′-end-labeled probes that were used for S1 nuclease mapping are shown at the bottom at the corresponding position. The stars indicate the positions of the radioactive label. Note that the probes are single-stranded cDNA fragments complementary to the ureB coding sequence and therefore oriented in the opposite direction from the ureB gene. (B) Scheme of the expected banding patterns in the S1 nuclease mapping experiments resulting from degradation of the intact mRNA of ureAB or transcription termination at a site downstream of the sRNA coding region. In the case of codegradation, both 3′- and 5′-end-labeled probes should detect low-molecular-weight bands corresponding to protection of the labeled probe by transcripts representing both the 3′ and 5′ ends of the degrading RNA, in addition to a band corresponding to the full-length transcript resulting from protection by the nondegraded mRNAs. In the case of termination, the 5′-end-labeled probe should detect only the full-length mRNA species that have not been terminated, while the 3′-end-labeled probe should detect a band corresponding to unterminated full-length mRNA and smaller distinct bands representing the 3′ end of the transcript up to the termination site. (C) Experimental results of the 5′- and 3′-end S1 nuclease mapping using a ³²P-labeled probe and total RNA from wild-type H. pylori (WT). A control experiment (probe) using the same probes only but without S1 nuclease treatment is also shown.