Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E

Abstract RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5′-sensor pocket that renders enzyme activity maximal on 5′-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself (‘recessive resurrection’). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5′-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5′-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH’s endonucleolytic action.


Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli
. RNase E-encoding plasmid variants constructed in this study Table S3. List of oligonucleotide primers Table S4. Growth rates (h -1 ) of ASKA plasmid-bearing derivatives of GJ15074 (rne + rne-lac recA) Figure S1. β-Galactosidase specific activity in rne-lac strains bearing Δrne or rne + on the chromosome, carrying ASKA plasmid vector or derivatives encoding different RNase E variants.     418Δ, I and G; and 395Δ, H and G. The primer pairs for truncations in the pRne-SG1 derivatives were: 493Δ, K and O; and 395Δ, L and G. When required, the Kan R or Cm R determinants (which are flanked by FRT sites) was excised with the aid of plasmid pCP20 as described (2), and the remnant scar is designated by the suffix '::FRT' in Supplementary Table S2. The excision experiments with pCP20 were successful even for determinants on the pRne-SG1 plasmid derivatives, despite the fact that both plasmids are pSC101 replicons.
The DNA sequence modifications required to generate the R169Q, D303A, or D346A substitutions in RNase E and its variants were introduced by site-directed mutagenesis of the plasmid-encoded genes. In each case, around 20-50 ng of plasmid DNA was used as template, with 20 pmol each of a complementary pair of oligonucleotide primers carrying the modification of interest. Following 30 cycles of "linear PCR" with Q5 High-Fidelity reaction mix (New England Biolabs, USA), the DNA preparation was treated with DpnI (to destroy the original plasmid DNA template) and transformed into DH5α.
Inheritance of the mutation was confirmed by DNA sequencing. The primer pairs used for the different substitutions were (primer sequences are given in Supplementary Table S3): R169Q, E and F; D303A, A and B; and D346A, C and D. The plasmids on which these substitutions were introduced included pASKA-rne, as also its derivative (pHYD5110) which was identified in this study to carry a spontaneous 15-bp deletion (of the sequence TATAATAGATTCAAT) in the T5-lac promoter region of the vector, resulting in reduced expression of RNase E even with 100 µM IPTG.

Generating rne truncations on the chromosome and pHYD1613
The modifications encoding RNase E truncations 493Δ, 438Δ, 418Δ and 395Δ were generated on the chromosome and on plasmid pHYD1613 by the recombineering method of Datsenko and Wanner (2), using a strain that carried rne + at both locations. The recombineering constructs were prepared by PCR on plasmid pKD13 as template, with the same primer pairs as described above for truncations on the pASKA plasmids. Following electroporation, Kan R colonies were selected and for each of the clones the site of Kan R insertion-deletion (whether on the chromosome or on pHYD1613) was determined through re-transformation experiments.

Other plasmid constructions
The lacƵ2526::Tn10dTet mutation was introduced into plasmid pHYD1613 and its rne-truncation derivatives by P1 transduction from strain GJ2278 (3). The ΔlacY::Kan mutation, sourced from the Keio collection (4), was also introduced into plasmid pHYD1613 by several sequential steps of P1 transduction. Plasmid pHYD5188 is a derivative of pET21b (Novagen, USA), and was constructed from plasmid pHYD3004 (5) by cloning an NdeI-XhoI PCR fragment encoding RNase E-395Δ downstream of the plasmid-borne T7 promoter, such that the encoded polypeptide carries a C-terminal His-tag; the primer pair used was M and N (Supplementary Table S3).

RT-qPCR experiments
Total RNA was extracted with Trizol reagent (Invitrogen) from cultures grown in LB to mid-exponential phase, and treated with RNase-free DNase I (Sigma). RNA integrity was checked by agarose gel electrophoresis (6). First strand cDNA synthesis was performed from 1 µg of RNA with randomhexamer primers, using RT and other reagents from, and protocol of, Maxima H kit (Thermo-Scientific). qPCR reactions and product quantitations were performed on an Applied Biosystems 7500 Real-time PCR System in a volume of 20 µl of SYBR Premix Ex Taq (TaKaRa).
Amplification of cDNA corresponding to part of the 5'-UTR of rne mRNA was achieved with the primer pair T and U, and likewise that for a part of 16S rRNA with the primer pair V and W (primer sequences are given in Supplementary Table S3). For every cDNA preparation and amplicon, at least three separate PCR experiments were done, each in duplicate: after heating for 5 min at 95°C, the mixtures were subjected to 40 cycles of amplification (95°C, 15 sec; 50°C, 1 min). Each of the primer pairs was associated with production of a single amplicon fragment of expected size (172 and 184 bp, respectively, for 5'-UTR and 16S rRNA regions), as determined by gel electrophoresis and melt-curve analysis. In control experiments, the RNA samples were also subjected to qPCR in absence of RT addition, and product yields were around 1000-fold less (˃ 9 cycles) than those in the RT-treated preparations.

Immunoblotting for RNase E and its variants
The procedure for immunoblotting was essentially as described by Sambrook and Russell (6). GJ15074 derivatives carrying the ASKA plasmids encoding full-length RNase E or its D303A (pHYD5152) substitution variant were grown to mid-exponential phase without or with 25 µM IPTG supplementation. GJ15074 with pCA24N vector, cultured with 50 µM IPTG, was taken as control. Around 10 9 cells were lysed in 200 µl of sodium dodecyl sulphate (SDS)-loading buffer at 95°C for 5 min, of which 20 µl was loaded for electrophoresis on a SDS-10% polyacrylamide gel. Separated proteins were electro-transferred to a polyvinylidene difluoride (PVDF) membrane by the wet transfer method (150 V, 90 min). The PVDF membrane was then stained with amido black.
All reagents used for immunodetection were from Sigma Chemicals, USA. After destaining, the PVDF membrane was placed in blocking solution (5% skimmed milk, 0.1% Tween 20, 150 mM NaCl, 20 mM Tris-Cl, pH 7.6) for 45 min; subsequently, the bound proteins on the membrane were reacted overnight at 4°C with mouse anti-His antibody at 1:10000 dilution, and then with secondary horseradish peroxidase-conjugated anti-mouse antibody at 1:10000 dilution for 1 h at room temperature. Immunoreactive bands on the blot were visualized with the aid of ECL solution kit in a chemiluminescence detection system.

Overexpression and purification of RNase E variants
MC4100 carrying the ASKA plasmid derivatives pHYD5183, pHYD5166, pHYD5182 or pHYD5181 (encoding, respectively, RNase E variants 493Δ, 395Δ, 395Δ-D303A and 395Δ-D346A, each with N-terminal His-tag) were grown overnight in 500 ml cultures supplemented with 0.1 mM IPTG. The variant 395Δ-R169Q with C-terminal His-tag was overexpressed from the pET21b-derived plasmid pHYD5188 in a 500 ml culture of the BL21(DE3) derivative with 0.1 mM IPTG for 2 h, essentially as earlier described (7). All steps of protein purification were done in presence of 1 M NaCl (8) All steps of protein purification were undertaken at 4°C. 1 ml of Ninitrilotriacetic acid (NTA) beads (GE Healthcare, USA) were washed with 5 ml of deionized water followed twice with 5 ml of lysis buffer. The beads were then incubated with the cell lysate supernatant on a low speed shaker for 1 h before they were poured to constitute a gravity column from which the flow-through was collected. The column was washed with 80 ml of wash buffer (20 mM Na phosphate pH 7.3, 1 M NaCl, 20 mM imidazole) before the bound protein was eluted in 10 ml of elution buffer (similar composition as wash buffer but with 500 mM imidazole).
The purified protein was subjected to buffer exchange with three changes of storage buffer (9) ( 1 L each, 20 mM Na phosphate pH 7.5, 200 mM NaCl), and then stored at -80°C in the same buffer with 20% glycerol. Protein estimations were performed with the aid of a commercial kit based on the bicinchoninic acid assay (10), and typical protein yields were around 0.5 mg for each of the proteins encoded by the ASKA plasmid derivatives and around 3 mg for 395Δ-R169Q.

Size-exclusion chromatography
The experiments of size-exclusion chromatography were performed at 4°C on   Education, India). l Prne-lac (also called lez1) has been described by Jain and Belasco (16).
GGTTACCTTGTTACGACTT a The nucleotide changes for site-directed mutagenesis are shown in bold. b NdeI site is italicized. c XhoI site is italicized, and sequence encoding His6-tag is underlined. ASKA-attenuated promoter ASKA-attenuated promoter ∆relA ∆spoT ∆rng Legend to Figure S2: Restoration of viability upon co-expression of pairs of lethal rne alleles (intragenic complementation). Description is as in legend to Figure 3. All strains carry ASKA plasmid derivatives with attenuated promoter (because of a 15-bp deletion) to express the RNase E variants and pHYD1613 (as shelter plasmid), and the plates were supplemented with 100 µM IPTG. Panels i-iv have employed strain GJ12087 (with plasmids pHYD5153, pHYD5149, pHYD5154, and pHYD5150 respectively for the four panels), along with pHYD2379 as the RNase E variant-encoding common second plasmid. The strain GJ15057 was used for panels v and vi with plasmids pHYD5154 and pHYD5150 respectively, once again along with pHYD2379 as the RNase E variant-encoding common second plasmid; these derivatives also carried the additional shelter plasmid pRC-SpoT, and the white colonies marked are those that have lost both shelter plasmids.   plasmids pHYD5152 and pHYD5151. The corresponding chromatogram for the rne + control plasmid pASKA-rne + is also shown. Sequence change from GAC to GCC, corresponding to change in the encoded amino acid from D to A, may be noted within the mauve-bordered window for the RNase E residues 303 and 346 in the corresponding mutants. The oligonucleotide primers used were (Supplementary Table S3): P and Q for PCR amplification from total cellular DNA, and R for sequencing. (B) Determination of RNase E expression levels in cultures of GJ15074 derivatives carrying pASKA-rne + or pHYD5152, after growth with 0 and 25 µM IPTG supplementation. GJ15074 with vector pCA24N, cultured with 50 µM IPTG, was used as a control. Following gel electrophoresis of the cell lysates, proteins were transferred to PVDF membrane and stained with amido black (left) or reacted with anti-His antibody (right). RNase E exhibits anomalous slow mobility during polyacrylamide gel electrophoresis (17), and its position of migration is marked by the arrow. Lane 1, molecular-weight standards; values (in kDa) for some of the bands on this lane are indicated in both panels. Figure S6 (... contd.)