Gastos de contingencias

In order to identify functional groups that are important for substrate cleavage by RNase P RNA, NAIM and NAIS experiments have to be performed with a self-cleaving RNA conjugate (P RNA - tRNA; Fig. 4.3.2). In this experimental setup, functional, self-cleaving conjugates can be separated from inactive ones according to size, employing denaturing PAGE (Harris & Pace, 1995). In this study two types of RNA conjugates were used:

P RNA – tRNA 5' -half

The P RNA – tRNA 5' -half conjugate (Fig. 4.3.2) is based on a transcript consisting of E. coli P RNA (black), a linker region (green) and the 5' -half of a bacterial tRNAGly (blue). To generate a functional substrate, an excess of tRNA 3' -half (red, Fig. 4.3.2) was annealed

to this construct, creating a self-cleaving P RNA – tRNA conjugate. This bipartite system prevents premature self-cleavage at the tRNA 5' -end during RNA preparation and furthermore enables NAIS-studies using modified functional groups in the 3' -half region of tRNA.

The sequence encoding the P RNA – tRNA 5' -half was constructed in three steps (Fig. 4.3.3). To create a homogenous end, a hammerhead sequence was attached to the 3' - end of the DNA construct; the hammerhead self – cleaves during RNA transcription.

(1) A standard PCR (3.3.4), using primers 1 (magenta) and 2 (green) and plasmid pFL 117 (encoding the sequence of P RNA from E. coli) as template;

(2) A primer elongation reaction (3.3.6) using primers 3 (blue) and 4 (red);

Fig. 4.3.2: E coli P RNA – tRNA conjugate; with the P RNA in black, linker in green, tRNA 5’ half in blue and 3’ half in red. The tRNA 3' -half is either only annealed to the 5' -half part of the conjugate (P RNA – tRNA 5' -half conjugate) or directly connected to the 5’ half (P RNA – tRNA conjugate).

Fig. 4.3.3: Construction of the P RNA – tRNA 5' -half conjugate; For simplification the primers have been numbered: primer 1 in magenta (Eco M1 5' Eco R I T7), 2 in green (3' M1 Gly spacer), 3 in blue (3' Gly spacer 2 compl) and 4 in red (3' Gly spacer 3). The sequence corresponding to each primer and their direction is indicated. Please note that the primer sequence is complementary to (primer 2 and 4) or identical with (primer 1 and 3) the given sequence (see also appendix 2). A part of the sequence not shown here is indicated by the broken line.

(3) A fill up reaction (3.3.7) using the double-stranded DNA products of step (1) and (2).

The DNA product obtained in step (3) was digested with Eco RI and Bam HI and ligated into the vector pUC 19 which was previously linearised using the same two restriction endonucleases and dephosphorylated at the 5’ end. E coli XL2 blue cells were transformed with the ligation product, and ampicillin resistant colonies were analysed (analytical plasmid preparation, restriction digest and sequencing [the latter being performed by MWG-Biotech]). The desired plasmid (called pXX) was isolated (3.3.1), linearised with Bam HI and subjected to in vitro transcription (3.4.1).

The tRNA 3' -half (Fig 4.3.2; red) encoding sequence was constructed by standard PCR (3.3.4), using the primers 9 (tgly 3’ -half 5' T7 EcoR I) and 10 (tgly 3’ -half 3' Mfe I- Bahm HI) and the plasmid pSBpt3’HH (encoding the ptRNAGly _{sequence) as template. The} PCR product was cloned into the vector pUC 19 using the restriction sites Eco RI and Bam HI. E coli XL2 blue cells were transformed with the ligation product, and ampicillin resistant colonies were analysed (see above). The desired plasmid (ptgly 3’ -half) was isolated (3.3.1), linearised with Mfe I (Mun I) and subjected to in vitro transcription (3.4.1). Linearisation with Mfe I enzyme ensured that CCA termini (important for P RNA catalysis) were generated during the run-off transcription.

P RNA – tRNA conjugate

The sequence encoding the P RNA–tRNA conjugate (Fig. 4.3.2) was constructed by PCR using as template the plasmid containing the P RNA–tRNA 5' -half sequence (pXX; see above). The PCR method applied in this case differed from the standard method (3.3.4) regarding the number and the amount of primers used (Fig. 4.3.4). Instead of one antisense primer, three overlapping ones were used in this special case, in order to generate the sequence for the tRNA 3' -half (which is not encoded by the template). The amount (1 nM) of

Fig. 4.3.4: Construction of the P RNA – tRNA conjugate; For simplification the primers have been numbered: primer 5 in light green (Eco M1 5' Eco RI T7 short), 6 in yellow (ptRNA loops), 7 in blue (3' ptRNA Mun I-Bam HI) and 8 in red (3' ptRNA short). The sequence corresponding to each primer and their orientation is indicated. Please note that the primer sequence is complementary to (primer 6, 7 and 8) or identical with (primer 5) the given sequence (see also appendix 2). A part of the sequence not shown here is indicated by the broken line.

short primers (5 in light green and 8 in violet; Fig. 4.3.4) added to the PCR mix corresponded to the standard PCR method (3.3.4). The concentration of the long primers (6 in yellow and 7 in dark blue; Fig. 4.3.4) was ten times lower.

The PCR product was cloned into the Eco RI and Bam HI sites of a pUC 19 derivative (pUC 19 Hind III to Eco 147I), containing a point mutation (introduced by PCR mutagenesis; 3.3.5) that transforms the Hind III recognition site into an Eco 147 I recognition site. The resulting plasmid was termed pXY. Absence of the Hind III site from the original vector was necessary for the construction of P RNA – tRNA conjugates with different linker lengths (Fig. 4.3.2; linker shown in green):

(1) The plasmid encoding the P RNA – tRNA conjugate sequence (pXY), containing a linker of 53 nt and constructed as described above, was digested with Hind III (2 recognition sites within the linker region) and religated, resulting in a conjugate with a linker of 33 nt;

(2) By annealing of a self – complementary deoxyoligonucleotide with 5’ phosphate termini, creating a recognition site for Eco 47 III (enzyme generating blunt ends) in the middle of the helix as well as appropriate overhangs, and ligating this short DNA duplex into the Hind III linearised vector [see (1)], a 43 nt linker was obtained;

(3) The plasmid obtained in (2) was linearised with Eco 47 III, and bluntend double - stranded DNA fragments of different size obtained from a 10 bp DNA ladder by separation on an agarose gel were ligated into the vector. The resulting products contained linkers between 54 and 375 nt.

For run - off transcription all templates, obtained as described above, were linearised with Mfe I (Mun I). Transcription was performed using the protocol for internally labelled RNA as described in 3.4.1 with one exception: the Mg2+ concentration was 7 instead of 33 mM. At this Mg2+ concentration, cis–cleavage of the conjugate during transcription was avoided.