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, Ng 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

4 Citations (Scopus)

Abstract

Escherichia coli RNase E contains a site that selectively binds to RNAs containing 5&πριμε;-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&πριμε; 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, 65HGFLPLK 71-which includes amino acids previously implicated in substrate binding and catalysis- and 24LYDLDIESP GHE QK 37-which includes the Q36 locus-were identified in wild-type enzyme complexes, whereas 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
JournalRNA Biology
Volume8
Issue number6
Publication statusPublished - 2011 Nov 1

Keywords

  • Q36R
  • RNA binding
  • RNA degradation
  • RNase E
  • RNase E regulation

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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  • Cite this

    Go, H., Moore, C. J., Lee, M., Shin, E., Jeon, C. O., Cha, N. J., Han, S. H., Kim, S. J., Lee, S-W., Lee, Y., Ha, N. C., Kim, Y. H., Cohen, S. N., & Lee, K. (2011). Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding. RNA Biology, 8(6).