Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding.

Hayoung Go, Christopher J. Moore, Minho Lee, Eunkyoung Shin, Che Ok Jeon, Chang Jun Cha, Seung Hyun Han, Su Jin Kim, Sang Won Lee, Younghoon Lee, Nam Chul Ha, Yong Hak Kim, Stanley N. Cohen, Kangseok Lee

Research output: Contribution to journalArticle

Abstract

Escherichia coli RNase E contains a site that selectively binds to RNAs containing 5'-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase E, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5' termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme's secondary or tertiary structure. Studies of cleavage of fluorogenic substrate and EMSA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. However, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart: two substrate-bound tryptic peptides, (65) HGFLPLK (71)--which includes amino acids previously implicated in substrate binding and catalysis--and (24) LYDLDIESPGHEQK (37)--which includes the Q36 locus-were identified in wild-type enzyme complexes. Only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase E locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects.

Original languageEnglish
Pages (from-to)1022-1034
Number of pages13
JournalRNA Biology
Volume8
Issue number6
Publication statusPublished - 2011 Nov 1
Externally publishedYes

Fingerprint

Up-Regulation
RNA
Mutation
Enzymes
Catalytic Domain
Binding Sites
RNA Cleavage
Catalytic RNA
Peptides
Glutamine
Catalysis
Fluorescent Dyes
Mutagenesis
Arginine
Mass Spectrometry
Phosphorylation
Escherichia coli
Amino Acids
ribonuclease E
Proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Go, H., Moore, C. J., Lee, M., Shin, E., Jeon, C. O., Cha, C. J., ... Lee, K. (2011). Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding. RNA Biology, 8(6), 1022-1034.

Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding. / Go, Hayoung; Moore, Christopher J.; Lee, Minho; Shin, Eunkyoung; Jeon, Che Ok; Cha, Chang Jun; Han, Seung Hyun; Kim, Su Jin; Lee, Sang Won; Lee, Younghoon; Ha, Nam Chul; Kim, Yong Hak; Cohen, Stanley N.; Lee, Kangseok.

In: RNA Biology, Vol. 8, No. 6, 01.11.2011, p. 1022-1034.

Research output: Contribution to journalArticle

Go, H, Moore, CJ, Lee, M, Shin, E, Jeon, CO, Cha, CJ, Han, SH, Kim, SJ, Lee, SW, Lee, Y, Ha, NC, Kim, YH, Cohen, SN & Lee, K 2011, 'Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding.', RNA Biology, vol. 8, no. 6, pp. 1022-1034.
Go H, Moore CJ, Lee M, Shin E, Jeon CO, Cha CJ et al. Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding. RNA Biology. 2011 Nov 1;8(6):1022-1034.
Go, Hayoung ; Moore, Christopher J. ; Lee, Minho ; Shin, Eunkyoung ; Jeon, Che Ok ; Cha, Chang Jun ; Han, Seung Hyun ; Kim, Su Jin ; Lee, Sang Won ; Lee, Younghoon ; Ha, Nam Chul ; Kim, Yong Hak ; Cohen, Stanley N. ; Lee, Kangseok. / Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding. In: RNA Biology. 2011 ; Vol. 8, No. 6. pp. 1022-1034.
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