Hayoung Go

All Publications

• Modulation of RNase E Activity by Alternative RNA Binding Sites. PloS one Kim, D., Song, S., Lee, M., Go, H., Shin, E., Yeom, J., Ha, N., Lee, K., Kim, Y. 2014; 9 (3)

  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.

  View details for DOI 10.1371/journal.pone.0090610

  View details for PubMedID 24598695

  View details for PubMedCentralID PMC3944109

• Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding RNA BIOLOGY Go, H., Moore, C. J., Lee, M., Shin, E., Jeon, C. O., Cha, C., Han, S. H., Kim, S., Lee, S., Lee, Y., Ha, N., Kim, Y., Cohen, S. N., Lee, K. 2011; 8 (6): 1022-1034

  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.

  View details for DOI 10.4161/rna.8.6.18063

  View details for Web of Science ID 000298630600012

  View details for PubMedID 22186084

  View details for PubMedCentralID PMC3360077

• 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 Shin, E., Go, H., Yeom, J., Won, M., Hanj, S. H., Bae, J., Han, S. H., Han, K., Lee, Y., Ha, N., Moore, C. J., Sohlberg, B., Cohen, S. N., Lee, K. 2008; 179 (4): 1871-1879

  Abstract

  RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing of a large fraction of RNAs in the cell. To better understand the molecular mechanisms of RNase E action, we performed a genetic screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that knock down the ability of RNase E to support survival of E. coli. Comparative phylogenetic analysis of RNase E homologs shows that wild-type residues at these mutated positions are nearly invariably conserved. Cells conditionally expressing these N-Rne mutants in the absence of wild-type RNase E show a decrease in copy number of plasmids regulated by the RNase E substrate RNA I, and accumulation of 5S ribosomal RNA, M1 RNA, and tRNA(Asn) precursors, as has been found in Rne-depleted cells, suggesting that the inability of these mutants to support cellular growth results from loss of ribonucleolytic activity. Purified mutant proteins containing an amino acid substitution in the DNase I subdomain, which is spatially distant from the catalytic site posited from crystallographic studies, showed defective binding to an RNase E substrate, p23 RNA, but still retained RNA cleavage activity-implicating a previously unidentified structural motif in the DNase I subdomain in the binding of RNase E to targeted RNA molecules, demonstrating the role of the DNase I domain in RNase E activity.

  View details for DOI 10.1534/genetics.108.088492

  View details for Web of Science ID 000258591200012

  View details for PubMedID 18660536

  View details for PubMedCentralID PMC2516065

• Inhibitory effects of RraA and RraB on RNAse E-related enzymes imply conserved functions in the regulated enzymatic cleavage of RNA FEMS MICROBIOLOGY LETTERS Yeom, J., Go, H., Shin, E., Kim, H., Han, S. H., Moore, C. J., Bae, J., Lee, K. 2008; 285 (1): 10-15

  Abstract

  RraA and RraB are recently discovered protein inhibitors of RNAse E, which forms a large protein complex termed the degradosome that catalyzes the initial step in the decay and processing of numerous RNAs in Escherichia coli. Here, we report that these E. coli protein inhibitors physically interact with RNAse ES, a Streptomyces coelicolor functional ortholog of RNAse E, and inhibit its action in vivo as well as in vitro; however, unlike their ability to differentially modulate E. coli RNAse E action in a substrate-dependent manner by altering the composition of the degradosome, both proteins appear to have a general inhibitory effect on the ribonucleolytic activity of RNAse ES, which does not interact with E. coli polynucleotide phosphorylase, a major component of the degradosome. Our findings suggest that these regulators of RNAse activity have a conserved intrinsic property enabling them to directly act on RNAse E-related enzymes and inhibit their general ribonucleolytic activity.

  View details for DOI 10.1111/j.1574-6968.2008.01205.x

  View details for Web of Science ID 000257658900002

  View details for PubMedID 18510556