Modulation of RNase E activity by alternative RNA binding sites

Abstract

Endoribonuclease E (RNase E) affects the composition and balance of the RNA population in Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (24LYDLDIESPGHEQK37) of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne) that was UV crosslinked with a 5'-32P-end-labeled, 13-nt oligoribonucleotide (p-BR13) containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (427LIEEEALK433), whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71). Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.

Figure 1. Identification of a hypoactive N-Rne mutant.

(A) Location of the isolated single amino-acid substitutions in the crystal structure of the N-terminal region of RNase E. Two tryptic peptides that were UV-crosslinked to p-BR13, ²⁴LYDLDIESPGHEQK³⁷ and ⁶⁵HGFLPLK⁷¹, are colored in blue and green, respectively. p-BR13 is colored in yellow. The diagram was generated using PyMOL software. (B) Tandem mass spectrum assigned to the predicted b- and y-ions generated by collision-induced fragmentation of the peptide, ²⁴LY^CDLDIESPGHEQK³⁷, with the Y25 residue bound to cytosine (m/z = 629.63, z = +3, mass error  = −1.20 ppm). (C) Tandem mass spectrum assigned to the predicted b- and y-ions generated from collision-induced fragmentation of the peptide, ²⁴LYDLDIESPGHEQ^AK³⁷, with the Q36 residue bound to adenine (m/z = 637.64, z = +3, mass error  = 8.62 ppm). (D) Growth characteristics of cells expressing wild-type N-Rne or the Q36R or Y25A mutant proteins. Growth of KSL2000 cells harboring pNRNE4, pNRNE4-Q36R, or pNRNE4-Y25A was measured individually on LB-agar plates containing 1.0 to 1000 µM IPTG. KSL2000 harboring pACYC177 grew only when full-length RNase E was expressed from pBAD-RNE in the presence of 0.2% arabinose. Numbers on the top indicate the number of bacterial cells in each spot.

Figure 2. Effects of Y25A and Q36R on the catalytic activity of RNase E in vivo and in vitro.

(A) Plasmid copy number of pNRNE4, pNRNE4-Q36R and pNRNE4-Y25A in KSL2000. Plasmids were purified from KSL2000 cells harboring pNRNE4, pNRNE4-Q36R or pNRNE4-Y25A and were digested with HindIII, which has a unique cleavage site in all of the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pBAD-RNE), which replicates independently of Rne, by measuring the molar ratio of the ColE1-type plasmid to the pBAD-RNE plasmid. (B) Growth characteristics of KSL2003 cells expressing wild-type N-Rne or the Q36R or Y25A mutant proteins. Growth of KSL2003 cells harboring pLAC-RNE2, pLAC-RNE2-Q36R, or pLAC-RNE2-Y25A was measured individually on LB-agar plates containing 1.0 to 1000 µM IPTG. Numbers on the top indicate the number of bacterial cells in each spot. (C) Plasmid copy number of pET28a in KSL2003. Plasmids were purified from KSL2003, KSL2003-Q36R or KSL2003-Y25A cells harboring pET28a and digested with HindIII, which has a unique cleavage site in all the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pLAC-RNE2, pLAC-RNE2-Q36R or pLAC-RNE2-Y25A) by measuring the molar ratio of the ColE1-type plasmid to the pSC101-derived plasmid. (D) Expression profiles of Rne and mutant proteins in KSL2003. The membrane probed with an anti-Rne polyclonal antibody was stripped and reprobed with an anti-S1 polyclonal antibody to provide an internal standard. The relative abundance of protein was quantified by setting the amount of wild-type Rne to 1. KSL2003 cells were grown in LB medium containing 10 µM IPTG. (E) In vitro cleavage of p-BR13 by wild-type N-Rne, Q36R and Y25A mutant proteins. Two pmol of 5′ end-labeled p-BR13 was incubated with 1 pmol of purified wild-type N-Rne or Q36R or Y25A mutant protein in 20 µl of cleavage buffer at 37°C. Samples were removed at each indicated time point and mixed with an equal volume of loading buffer. Samples were denatured at 65°C for 5 min and loaded onto 15% polyacrylamide gel containing 8 M urea. The radioactivity in each band was quantified using a phosphorimager and OptiQuant software.

Figure 3. Effects of mutant proteins on RNA binding activity.

(A, B) Electrophoretic mobility shift assay. The 5′ end labeled p-BR13 (0.5 pmol) was incubated with increasing concentrations of purified wild-type N-Rne or Q36R or Y25A mutant protein in 20 µl of EMSA buffer, incubated on ice (A) or at room temperature (B) for 10 min, and analyzed by 12% nondenaturing PAGE. Binding constants were calculated based on slopes calculated from the graph. To avoid induction of RNA cleavage, Mg²⁺ was omitted from the EMSA reactions. (C) UV crosslinking of N-Rne-wt, N-Rne-Q36R and N-Rne-Y25A to p-BR13. Two pmol of p-BR13 was incubated with 100 pmol of N-wild-type Rne, Q36R or Y25A mutant protein in 20 µl of crosslinking buffer and exposed to UV light for 30 min. Samples were loaded onto 10% polyacrylamide gels (lanes 2, 5, 8) and samples in the absence of p-BR13 (lane 1, 4, 7) or UV irradiation (lanes 3, 6, 9) were also loaded as controls. The gel was stained with Coomassie brilliant blue and dried. The radioactivity in each band was detected using a phosphorimager and OptiQuant software. The number of crosslinked p-BR13 per pmol of protein was calculated.

Figure 4. Mass spectrometry analysis of wild-type and mutant N-Rne proteins obtained from UV-crosslinking.

(A) Partial peptide sequence of the wild-type N-Rne showing the regions of an internal standard (IS) sequence and nucleoside-bound peptides, denoted as R, P, and M sites, corresponding to an uncompetitive site (a.a. 26–37), a catalytic site (a.a. 65–71) and an allosteric site (a.a. 427–433), respectively. (B) Extracted ion chromatograms (XICs: panels a, c and e) and corrected PSM levels (panels b, d and f) of nucleoside-bound peptides and their parent peptides of wild-type N-Rne (a and b) and Q36R (c and d) and Y25A (e and f) mutants. Nucleoside-bound peptide peaks in the XICs are denoted with asterisks to the right of the symbols, R, P, and M, corresponding to the trypsin/chymotrypsin-digested peptides, ²⁶DLDIESPGHEQK³⁷, ⁶⁵HGFLPLK⁷¹, and ⁴²⁷LIEEEALK⁴³³, respectively, in the sequence of the wild-type N-Rne. The Y25A and Q36R mutants replace the R sequence with ²⁴LADLDIESPGHEQK³⁷ and ²⁶DLDIESPGHER³⁶, respectively. In the right panels, the numbers of peptide spectrum matches (PSMs) of the parental and nucleoside-bound peptides are shown in parentheses above the black and gray bars of the corrected PSM levels. The relative levels of internal standard (IS), ¹⁶VALVDGQR²³, are expressed as 100 and one unit for the calculation of relative intensity of XIC and correction of PSM levels, respectively. Tandem mass spectrometry data are given in Table S1 and Figure S2 in File S1.