Table 1.
Plasmids
| Name | Relevant features/genotype | References |
|---|---|---|
| pIK163 | pBR322 carrying ecoRIRM operon | (10) |
| pHSG415 | pSC101 derivative, thermo-sensitive replication, KanR ApR, CmR | (41) |
| pIK172 | pHSG415 carrying the WT ecoRIRM, ApR, CmR | (11) |
| pIK173 | pHSG415carrying ecoRIR–M+, ApR, CmR | (11) |
| pGEM-T | E. coli TA cloning vector, ApR | Promega |
| pGEM-T2 | pGEM-T carrying an ecoRIRM operon fragment (+431 – +612) | (29) |
| pGEM-T4 | pGEM-T carrying an ecoRIRM operon fragment (+536 – +777) | (29) |
| pLY2 | pACYC184 carrying promoter-less lacZ gene | (29) |
| pLY66 | Transcriptional fusion of a WT ecoRIRM operon fragment with PR promoter (−67 to +400) fused to lacZ in pLY2 | (29) |
| pBLY18 | pJFY161 derivative, double mutations in −10 boxes of PM1M2 promoters (TATAAT to TAGCGG and TATATT to TAAAGC) | This study |
| pJFY35 | Transcriptional fusion of a WT ecoRIRM operon fragment (+873 to +660) to lacZ in pLY2 | This study |
| pJFY47 | Transcriptional fusion of a WT ecoRIRM operon fragment (+873 – +474) to lacZ in pLY2 | This study |
| pJFY153 | Transcriptional fusion of a WT ecoRIRM operon fragment (+873 to +535) to lacZ in pLY2 | This study |
| pJFY154 | Transcriptional fusion of the WT ecoRIRM operon fragment (+873 to +676) to lacZ in pLY2 | This study |
| pJFY185 | Transcriptional fusion of a WT ecoRIRM operon fragment (+873 – +633) to lacZ in pLY2 | This study |
| pJFY161 | Transcriptional fusion of a WT ecoRIRM operon fragment (+873 – +585) to lacZ in pLY2 | This study |
| pJ18 | pJFY154 derivative, up mutations in the −10 and −35 boxes of PREV0 (TATGAT→TATAAT; TTGTAG to TTGACA) | This study |
| pJ20 | pJFY154 derivative, down mutations in the −10 box of PREV0 (TATGAT→CCCGGG) | This study |
| pIM13 | Transcriptional fusion of a WT ecoRIRM operon fragment with promoter PM1,M2 (+585 to +784) fused to lacZ in pLY2 | This study |
| pIM15 | Transcriptional fusion of a WT ecoRIRM operon fragment with promoter PM1,M2 (+406 to +873) fused to lacZ in pLY2 | This study |
| pIM16 | Transcriptional fusion of a ecoRIRM operon fragment with PM1/PM2 promoter (+406 to +709) fused to lacZ in pLY2; PREV0 promoter hexamers are not present | This study |
| pIM18 | pJFY154 derivative, mutations in the −35 box of PREV0 (TTGTAG→GGCTAG) | This study |
| pIM19 | pJFY154 derivative, mutations in the −10 and −35 boxes of PREV0 (TATGAT→TGTGGT and TTGTAG→TTGTAA); no change in R.EcoRI amino-acid sequence | This study |
| pIM11 | Transcriptional fusion of a WT ecoRIR fragment with PR and PM1,M2 promoters (−67–+784) fused to lacZ in pLY2 | This study |
| pIM21 | Derivative of pIM11, mutation as in pJ20 to inactivate PREV0 promoter | This study |
| pIM30 | Derivative of pIM11, mutations as in pBLY18 to inactivate PM1,M2 promoter | This study |
| pIMRM | Entire WT EcoRI system in pACYC184 backbone; CmR | This study |
| pIM24 | pIMRM derivative, 3-nt substitutions as in pIM19 in the −10 and −35 boxes of PREV0 (TATGAT→TGTGGT and TTGTAG→TTGTAA); no change in R.EcoRI amino acid sequence | This study |
| pIM27 | pIMRM derivative; R–M+, deletion of HindIII–BglII fragment of ecoRIR | This study |
| pIM-REV0 | pUC18 derivative carrying ecoRIA gene for the antisense RNA (Rna0) from PREV0 promoter; 4-nt up mutations in PM1,M2 reverse promoter (TGGAAG→GGGATG; TTGTTA→AAGTTA) | This study |
| pIM-ΔRNA | pUC18 derivative with a part of ecoRIR gene (−67 – +130), used as a negative control for testing antisense RNA | This study |
R, restriction; M, modification; WT, wild-type; Cm, chloramphenicol; Ap, ampicillin; Kan, kanamycin.