3 Methods
3.2 Cloning
3.2.5 Plasmids generated throughout this study
3.2.5.1 Plasmids for complementation assays
Plasmids used for complementation assays contain a P RNA gene, or a P RNA gene plus a P protein gene, as well as their promoters and sometimes also terminators. In general, these plasmids are derivatives of pSP64 (Promega), pHY300 (Ishiwa and Shibaharasone 1986), pBsrnpAnHis (also called pB.s.[rnpA-NH+rnpB]) (Gössringer et al. 2006) and pACYCmitEcm1ApaI (also named pACYC177 E. coli rnpBwt) (Wegscheid and Hartmann 2006).
pSP64 variants
The pSP64 variants were constructed for RNase P RNA complementation assays in E. coli strain DW2.
● pSP64-Cpa rnpB-ApaI
The vector pSP64 was prepared by cutting pSP64_ecoli_m1_ApaI with Apa I and BamH I. In plasmid pSP64_ecoli_m1_ApaI, the E. coli rnpB gene was under the control of the native E.
coli rnpB promoter as described (Hardt and Hartmann 1996). The C. paradoxa P RNA gene was amplified from pUC19-Cpa wt, also called pT7G3CyRPR (Cordier and Schön 1999), with primers 5\’ApaI Cyp rnpB and 3\’Cyp rnpB BamHI; the PCR product was cut by Apa I and BamH I and then ligated into the vector using T4 DNA ligase. The C. paradoxa P RNA gene thus replaced E. coli rnpB, and was still under the control of the native E. coli rnpB promoter.
5\’ApaI Cyp rnpB
5’-CCAGGGCCCGCAAACCCTCTATACTGCGCGCCAAACGAATTTAATTAATGATT 3\’Cyp rnpB BamHI
5’-GCGGGATCCAAACGAACTTAATTTTAAGC
● pSP64-Cpa rnpB C57/G213-ApaI
The same cloning strategy was used as for pSP64-Cpa rnpB-ApaI. The C. paradoxa P RNA gene mutant C57/G213 was amplified from pT7G3CyRPR-C57/G213 (Cordier and Schön 1999) with primers 5\’ApaI Cyp rnpB and 3\’Cyp rnpB BamHI and introduced into pSP64_ecoli_m1_ApaI instead of E. coli rnpB.
● pSP64-Cpa rnpB C57/G213/G22-ApaI
Again the same cloning strategy was used as for pSP64-Cpa rnpB-ApaI. The C. paradoxa P RNA gene mutant C57/G213/G22 was amplified from pT7G3CyRPR-C57/G213/G22 (Cordier and Schön 1999) using primers 5\’ApaI Cyp rnpB and 3\’Cyp rnpB BamHI and introduced into the pSP64 vector instead of E. coli rnpB.
● pSP64-Mth rnpB-ApaI
The cloning strategy was as for pSP64-Cpa rnpB-ApaI. The M. thermoautotrophicus P RNA gene was amplified from pUC119_T7_M.th._rnpB (Jim Brown, North Carolina State University) using primers 5\’ApaI Mth rnpB and 5\’Mth rnpB BamHI and introduced into the pSP64 vector instead of E. coli rnpB.
5\’ApaI Mth rnpB
5’-CCAGGGCCCGCAAACCCTCTATACTGCGCGCCAGCCGAAGGGCAGCTGA 5\’Mth rnpB BamHI
5’-GCGGGATCCTGCCGAGAGTAACCCACCT
pHY300 variants
The shuttle vector pHY300 can replicate in E. coli cells and also B. subtilis cells. In the pHY300 derivatives used in this study, variants of P RNA genes were inserted into pHY300 for RNase P RNA complementation assays in the B. subtilis strain SSB318.
● pHY300-EE
The plasmid pHY300 was cut by Xba I and Hind III. The E. coli rnpB gene and its promoter were cut from pSP64_ecoli_m1_ApaI by Nhe I and Hind III and inserted in pHY300 via ligation by T4 DNA ligase.
● pHY300-Cpa rnpB wt
The same cloning strategy as for pHY300-EE was applied. The C. paradoxa P RNA gene and its promoter were cut from pSP64-Cpa rnpB-ApaI by Nhe I and Hind III and inserted in pHY300.
● pHY300-Cpa rnpB C57/G213
As above, The C. paradoxa P RNA gene mutant C57/G213 and its promoter were cut from pSP64-Cpa rnpB C57/G213-ApaI by Nhe I and Hind III and inserted in pHY300.
● pHY300-Cpa rnpB C57/G213/G22
Site-directed mutagenesis was performed to introduce G22 (underlined) into pHY300-Cpa rnpB C57/G213 with primers G22-Cyp-rnpB1 and G22-Cyp-rnpB2.
G22-Cyp-rnpB1
5’-CGAATTTAATTAATGATTGCAGATTTATTCAATCTGAGG G22-Cyp-rnpB2
5’-CCTCAGATTGAATAAATCTGCAATCATTAATTAAATTCG
● pHY300-EC
A megaprimer mutagenesis was done to change the S-domain coding region of E. coli rnpB into that of C. paradoxa as encoded on pHY300-Cpa rnpB wt. The megaprimer was generated from pHY300-EE with primers 119 and 120.
119 C. p. specificity s
5’-GTCCGGGCTCCATAGGGCAGAATTGCTGGGTAATTCCCAG 120 C. p. specificity as
5’-CCTATTTGGCCTTGCTCCGAACGGGGTTTACTAAGTAT
● pHY300-CE
First two PCR fragments were produced from the C. paradoxa rnpB gene. One was generated with primers 113 and 114 and contained the 5’-part of the C. paradoxa C-domain. The other was obtained with primers 115 and 116 and contained the C-domain’s 3’-part. The primers introduced additional restriction enzyme cleavage sites, a T7 promoter and nucleotides matching the 5’- or 3’- termini of the E. coli rnpB S-domain. After gel purification the two PCR products were used as primers for amplification of the E. coli rnpB S-domain. Then the final PCR product was cloned into pUC19 via BamH I and EcoR I restriction sites. A site-directed mutagenesis was done to complete the preparation of the chimeric rnpB gene CE with primers 143 and 144, which was amplified with primers 5\’ApaI Cyp rnpB plus 140 and inserted into pHY300-Cpa rnpB wt cut by Xba I and Apa I.
113
5’-GCGGGATCCtaatacgactcactatagCGAATTTAATTAATGATTACAG
114
5’-CAGGCGTTACCTGGCACCCTAACCTTATAGGAGCC 115
5’-GTGGCACGGTAAACTCCACCCAGGAGCAAAGTTTAGCG 116
5’-GCGGAATTCAAAACGAACTTAATTTTAAGCC 143 M Cypcat-Ecolispec s
5’-CCTGGCACCCTAACCTTTAAGGAGCCCGGACTTTCC 144 M Cypcat-Ecolispec as
5’-GGAAAGTCCGGGCTCCTTAAAGGTTAGGGTGCCAGG 5\’ ApaI Cyp rnpB
5’-CCAGGGCCCGCAAACCCTCTATACTGCGCGCCAAACGAATTTAATTAATGATT 140 XbaI-Cpy cat
5’-GCGTCTAGAGGATCCAAACGAACTTAATTTTAAGC
● pHY300-MM
Cloning was similar to pHY300-EE. The M. thermoautotrophicus P RNA gene and its promoter were excised from pSP64-Mth rnpB-ApaI by Nhe I and Hind III and inserted into pHY300.
● pHY300-EM
This plasmid was constructed by megaprimer mutagenesis on the basis of pHY300-EE. The megaprimer was generated by several PCRs. PCR I was performed to amplify the S-domain of the M. thermoautotrophicus P RNA gene with primers 125 plus 126 and template pHY300-MM. Then PCR II was done to combine the 5’ part of the E. coli rnpB C-domain with the S-domain of M. thermoautotrophicus P RNA gene, using templates pHY300-EE and the PCR I product for overlap extension and primers 100 and 126 for amplification of the product. In parallel, the combination of the M. thermoautotrophicus S-domain with the 3’ part of the C-domain of E. coli rnpB was obtained by PCR III using templates pHY300-EE and PCR I product for overlap extension and primers 99BWegscheid plus 125 for amplification of the product. Then the PCR II and III products were combined for overlap extension, and the extension product was amplified with primers 100 and 99BWegscheid to produce the megaprimer.
125 M.th. specificity s
5’-GTCCGGGCTCCATAGGGCACCGTGGTGCCGTGAGGCAT 126 M.th. specificity as
5’-CCTATTTGGCCTTGCTCCCCGTGGAATGGCCGTTTCAC 100 3 Chim-XbaI 2
5’-CGACCTGCAGATCTCTAGATTGCTGCTCAAGAACAGC 99 BWegscheid
5’-GCCAGGGGGAAACGCC
● pHY300-ME
First the S-domain of E. coli rnpB was amplified from pHY300-EE with primers 123 and 124 (PCR I). Then two PCRs were separately done to produce (i) the 5’ part of the M.
thermoautotrophicus C-domain plus E. coli S-domain (primers: 100 and124; templates:
pHY300-MM and PCR I product) and (ii) E. coli S-domain plus the 3’ part of the M.
thermoautotrophicus C-domain (primers: 123 and 99BWegscheid; templates: pHY300-MM and PCR I product). With primers 100 plus 99BWegscheid and the latter two PCR products as overlap extension templates, the large DNA segment containing the chimeric P RNA gene ME was
generated. Vector pHY300 and this large DNA segment were both cut with Xba I and then ligated to yield the recombinant plasmid. Because of an additional C accidently introduced in the middle of primer 124, a further site-directed mutagenesis with primers 138 and 139 was performed to delete the C residue.
123 M.th. catalytic s
5’-ACTCCACCCATCATACAGAAGGGTGCCAGGTAACGCCTG 124 M.th. catalytic as
5’-CAGCATTTGTCCTTGCATCCGGGTGGAGTTTACCGTGCCA 100 3 Chim-XbaI 2
5’-CGACCTGCAGATCTCTAGATTGCTGCTCAAGAACAGC 99BWegscheid
5’-GCCAGGGGGAAACGCC 138 Mth cat as (124 new)
5’-CAGCAT TTGTCCTTGCATC∆GGGTGGAGTTTACCGTGCCA 139 s138
5’-TGGCACGGTAAACTCCACCC∆GATGCAAGGACAAATGCTG
● pHY300-Ecat
Using pHY300-EE as template, an “inside-out”-PCR mutagenesis was done to remove the S-domain of E. coli P RNA with primers 145 and 146. Helix P7 of the S-S-domain was retained within this construct and was capped with a GAAA tetraloop (underlined).
145 Ecoli cat alone s
5’-GAAACACCCGGAGCAAGGCCA 146 Ecoli cat alone as
5’-CACCCTGCCCTATGGAGCC
pBsrnpAnHis derivative
pBsrnpAnHis, also called pB.s.[rnpA-NH+rnpB] (Gössringer et al. 2006), contains a B.
subtilis rnpA gene, encoding the RNase P protein, and a B. subtilis rnpB gene. Its derivative was produced for RNase P holoenzyme complementation assays also in B. subtilis strain SSB318.
● pBsrnpAnHis_ME-mJ15/18/2/3/P2/nP1
This plasmid was constructed by replacing the B. subtilis rnpB in pBsrnpAnHis with the mutant chimeric ME-J15/18/2/3/P2/nP1 P RNA gene. The first PCR was done to amplify the
mutated M. thermoautotrophicus P RNA gene from pUC19-ME-mJ15/18/2/3/P2/nP1 with primers 275 and Mth-nP1-3. The second PCR copied the B. subtilis native Rho-independent rnpB terminator from pBsrnpAnHis with primers 45BWegscheid and 276. Then the two PCR products were combined for overlap extension in a third PCR using primers 275 and 276 for amplification to generate a fusion of the mutated M. thermoautotrophicus P RNA gene and the B. subtilis rnpB terminator. The final PCR product was digested with Hind III and ligated into pBsrnpAnHis cut with Hind III and Swa I. A fortuitous mutation was corrected by site-directed mutagenesis with primers 285 and 286.
275 HindIII-ME
5’-CAGCAAGCTTCCGGGCAAGCCGAAGGGCAG Mth-nP1-3 (designed by M. Gössringer)
5’-GCTTGTTTTCATCATTTTAAATGTCCGGGCATGCCGAGAGTAACCCACCTTCTG 45BWegscheid
5’-ACATTTAAAATGATGAAAACAAGC 276 terminator B
5’-AAATTCTTTTCAAGATAAAAGCTC 285 A-G a
5’-CGAAACAGAAGGTGGGTTACTCTCGGCATGCCCGGACATTTAAAATG 286 A-G b
5’-CATTTTAAATGTCCGGGCATGCCGAGAGTAACCCACCTTCTGTTTCG
● pACYC-ME-mJ15/18/2/3/P2/nP1
A derivative of pACYCmitEcm1ApaI, also named pACYC177 E. coli rnpBwt (Wegscheid and Hartmann 2006), was generated for RNase P holoenzyme complementation assays in E.
coli strain BW. Towards this goal, the mutant ME-mJ15/18/2/3/P2/nP1 P RNA gene was amplified from pUC19-ME-mJ15/18/2/3/P2/nP1 with primers 277 plus 278 and ligated into pACYCmitEcm1ApaI, cut with Apa I and Xba I.
277 ApaI-ME
5’-CGCGGGCCCGCAAACCCTCTATACTGCGCGCCCCGGGCAAGCCGAAGGGCAG 278 XbaI-ME
5’-CGCTCTAGAGGATCCCCGGGCATGCCGAGAGTAACCCACCTTCTG