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.
References
[1]
Celesnik H, Deana A, Belasco JG (2007) Initiation of RNA decay in Escherichia coli by 5′ pyrophosphate removal. Mol Cell 27: 79–90. doi: 10.1016/j.molcel.2007.05.038
[2]
Kime L, Jourdan SS, Stead JA, Hidalgo-Sastre A, McDowall KJ (2009) Rapid cleavage of RNA by RNase E in the absence of 5′ monophosphate stimulation. Mol Microbiol 76: 590–604. doi: 10.1111/j.1365-2958.2009.06935.x
[3]
McDowall KJ, Kaberdin VR, Wu SW, Cohen SN, Lin-Chao S (1995) Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops. Nature 374: 287–290. doi: 10.1038/374287a0
[4]
Murashko ON, Kaberdin VR, Lin-Chao S (2012) Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity. Proc Natl Acad Sci U S A 109: 7019–7024. doi: 10.1073/pnas.1120181109
[5]
Kido M, Yamanaka K, Mitani T, Niki H, Ogura T, et al. (1996) RNase E polypeptides lacking a carboxyl-terminal half suppress a mukB mutation in Escherichia coli. J Bacteriol 178: 3917–3925.
[6]
McDowall KJ, Cohen SN (1996) The N-terminal domain of the rne gene product has RNase E activity and is non-overlapping with the arginine-rich RNA-binding site. J Mol Biol 255: 349–355. doi: 10.1006/jmbi.1996.0027
[7]
Koslover DJ, Callaghan AJ, Marcaida MJ, Garman EF, Martick M, et al. (2008) The crystal structure of the Escherichia coli RNase E apoprotein and a mechanism for RNA degradation. Structure 16: 1238–1244. doi: 10.1016/j.str.2008.04.017
[8]
Mackie GA (2013) RNase E: at the interface of bacterial RNA processing and decay. Nat Rev Microbiol 11: 45–57. doi: 10.1038/nrmicro2930
[9]
Callaghan AJ, Aurikko JP, Ilag LL, Grossmann JG, Chandran V, et al. (2004) Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E. J Mol Biol. 340: 965–979. doi: 10.1016/j.jmb.2004.05.046
[10]
Go H, Moore CJ, Lee M, Shin E, Jeon CO, et al. (2011) Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding. RNA Biol 8: 1022–1034. doi: 10.4161/rna.8.6.18063
[11]
Lee K, Cohen SN (2003) A Streptomyces coelicolor functional orthologue of Escherichia coli RNase E shows shuffling of catalytic and PNPase-binding domains. Mol Microbiol 48: 349–360. doi: 10.1046/j.1365-2958.2003.03435.x
[12]
Mudd EA, Higgins CF (1993) Escherichia coli endoribonuclease RNase E: autoregulation of expression and site-specific cleavage of mRNA. Mol Microbiol 9: 557–568. doi: 10.1111/j.1365-2958.1993.tb01716.x
[13]
Jain C, Belasco JG (1995) Autoregulation of RNase E synthesis in Escherichia coli. Nucleic Acids Symp Ser 85–88.
[14]
Sousa S, Marchand I, Dreyfus M (2001) Autoregulation allows Escherichia coli RNase E to adjust continuously its synthesis to that of its substrates. Mol Microbiol 42: 867–878. doi: 10.1046/j.1365-2958.2001.02687.x
[15]
Khemici V, Poljak L, Luisi BF, Carpousis AJ (2008) The RNase E of Escherichia coli is a membrane-binding protein. Mol Microbiol 70: 799–813. doi: 10.1111/j.1365-2958.2008.06454.x
[16]
Liou GG, Jane WN, Cohen SN, Lin NS, Lin-Chao S (2001) RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribonuclease E. Proc Natl Acad Sci U S A 98: 63–68. doi: 10.1073/pnas.98.1.63
[17]
Lee K, Zhan X, Gao J, Qiu J, Feng Y, et al. (2003) RraA. a protein inhibitor of RNase E activity that globally modulates RNA abundance in E. coli. Cell 114: 623–634. doi: 10.1016/s0092-8674(03)00646-9
[18]
Gao J, Lee K, Zhao M, Qiu J, Zhan X, et al. (2006) Differential modulation of E. coli mRNA abundance by inhibitory proteins that alter the composition of the degradosome. Mol Microbiol 61: 394–406. doi: 10.1111/j.1365-2958.2006.05246.x
[19]
Gorna MW, Pietras Z, Tsai YC, Callaghan AJ, Hernandez H, et al. (2010) The regulatory protein RraA modulates RNA-binding and helicase activities of the E. coli RNA degradosome. RNA 16: 553–562. doi: 10.1261/rna.1858010
[20]
Lee K, Bernstein JA, Cohen SN (2002) RNase G complementation of rne null mutation identifies functional interrelationships with RNase E in Escherichia coli. Mol Microbiol 43: 1445–1456. doi: 10.1046/j.1365-2958.2002.02848.x
[21]
Eng JK, McCormack AL, Yates JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5: 976–989. doi: 10.1016/1044-0305(94)80016-2
[22]
Tamura M, Lee K, Miller CA, Moore CJ, Shirako Y, et al. (2006) RNase E maintenance of proper FtsZ/FtsA ratio required for nonfilamentous growth of Escherichia coli cells but not for colony-forming ability. J Bacteriol 188: 5145–5152. doi: 10.1128/jb.00367-06
[23]
Lin-Chao S, Wong TT, McDowall KJ, Cohen SN (1994) Effects of nucleotide sequence on the specificity of rne-dependent and RNase E-mediated cleavages of RNA I encoded by the pBR322 plasmid. J Biol Chem 269: 10797–10803.
[24]
Shin E, Go H, Yeom JH, Won M, Bae J, et al. (2008) Identification of amino acid residues in the catalytic domain of RNase E essential for survival of Escherichia coli: functional analysis of DNase I subdomain. Genetics 179: 1871–1879. doi: 10.1534/genetics.108.088492
[25]
Yeom JH, Lee K (2006) RraA rescues Escherichia coli cells over-producing RNase E from growth arrest by modulating the ribonucleolytic activity. Biochem Biophys Res Commun 345: 1372–1376. doi: 10.1016/j.bbrc.2006.05.018
[26]
Urlaub H, Holsken EK, Luhrmann R (2008) Analyzing RNA-protein crosslinking sites in unlabeled ribonucleoprotein complexes by mass spectrometry. Methods Mol Biol 488: 221–245. doi: 10.1007/978-1-60327-475-3_16
Goldblum K, Apirion D (1981) Inactivation of the ribonucleic acid-processing enzyme ribonuclease E blocks cell division. J Bacteriol 146: 128–132.
[29]
Chang AC, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134: 1141–1156.
