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

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

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

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

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, Ng 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.

Research output: Contribution to journalArticle

Go, H, Moore, CJ, Lee, M, Shin, E, Jeon, CO, Cha, NJ, Han, SH, Kim, SJ, Lee, S-W, 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.
Go, Hayoung ; Moore, Christopher J. ; Lee, Minho ; Shin, Eunkyoung ; Jeon, Che Ok ; Cha, Ng 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.
@article{a65a28a120bf44758e893fef1d24552a,
title = "Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding",
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.",
keywords = "Q36R, RNA binding, RNA degradation, RNase E, RNase E regulation",
author = "Hayoung Go and Moore, {Christopher J.} and Minho Lee and Eunkyoung Shin and Jeon, {Che Ok} and Cha, {Ng Jun} and Han, {Seung Hyun} and Kim, {Su Jin} and Sang-Won Lee and Younghoon Lee and Ha, {Nam Chul} and Kim, {Yong Hak} and Cohen, {Stanley N.} and Kangseok Lee",
year = "2011",
month = "11",
day = "1",
language = "English",
volume = "8",
journal = "RNA Biology",
issn = "1547-6286",
publisher = "Landes Bioscience",
number = "6",

}

TY - JOUR

T1 - Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

AU - Go, Hayoung

AU - Moore, Christopher J.

AU - Lee, Minho

AU - Shin, Eunkyoung

AU - Jeon, Che Ok

AU - Cha, Ng Jun

AU - Han, Seung Hyun

AU - Kim, Su Jin

AU - Lee, Sang-Won

AU - Lee, Younghoon

AU - Ha, Nam Chul

AU - Kim, Yong Hak

AU - Cohen, Stanley N.

AU - Lee, Kangseok

PY - 2011/11/1

Y1 - 2011/11/1

N2 - 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.

AB - 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.

KW - Q36R

KW - RNA binding

KW - RNA degradation

KW - RNase E

KW - RNase E regulation

UR - http://www.scopus.com/inward/record.url?scp=81255197805&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=81255197805&partnerID=8YFLogxK

M3 - Article

C2 - 22186084

AN - SCOPUS:81255197805

VL - 8

JO - RNA Biology

JF - RNA Biology

SN - 1547-6286

IS - 6

ER -