Substrate-dependent effects of quaternary structure on RNase E activity.

RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5' terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.

Modulation of RNase E activity by alternative RNA binding sites.

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.

Metal-particle-induced enhancement of the photoluminescence from biomolecule-functionalized carbon nanotubes.

The effect of metal particles on the photoluminescence (PL) and the Raman spectra of functionalized SWCNTs in aqueous solutions was systematically investigated by studying three different metal particles (gold, cobalt, and nickel) on three different SWCNT suspensions (DNA-, RNA-, and sodium deoxycholate salt (DOC)-functionalized SWCNTs). Substantial enhancement of the PL intensities was observed, while the Raman spectra remained unchanged, after gold, cobalt, or nickel particles were introduced into RNA-SWCNT aqueous suspensions. Almost the same results were obtained after the same metal particles were added to DNA-SWCNT aqueous suspensions. However, both the PL and the Raman spectra did not exhibit any change at all after the same metal particles were introduced into DOC-SWCNT aqueous suspensions. The unusual PL enhancements observed in this work cannot be accounted for by the three well-known mechanisms in the literature: surface-enhanced Raman scattering effect, Förster resonance energy transfer in a rebundling of isolated SWCNTs, and pH changes of the aqueous solutions.

2-nitrobenzoate 2-nitroreductase (NbaA) switches its substrate specificity from 2-nitrobenzoic acid to 2,4-dinitrobenzoic acid under oxidizing conditions.

2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant Δ65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Δ193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5'-dithio-bis-(2-nitrobenzoic acid) and ZnCl(2), which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H(2)O(2). SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions.

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

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.

A genetic system for RNase E variant-controlled overproduction of ColE1-type plasmid DNA.

Escherichia coli endoribonuclease RNase E (Rne) regulates replication of ColE1-type plasmids by cleaving RNA I transcripts, which are synthesized from the plasmid and regulate the plasmid replication as antisense RNA. Here, we report the development of a genetic system that efficiently overproduces ColE1-type plasmid DNA when an RNase E variant that confers a hyperactive phenotype in RNA I cleavage is conditionally expressed from chromosome. This genetic system offers a method for isolation of large quantities of pure ColE1-type plasmid DNA, which have been most commonly used as molecular biology and biotechnology tools for research and industrial purposes.

Modulation of biological processes in the nucleus by delivery of DNA oligonucleotides conjugated with gold nanoparticles.

The development of a method that can efficiently deliver nucleic acids into the nucleus of living systems remains one of the key challenges for experimental and therapeutic use of nonbiological gene delivery agents. In the current study, we demonstrate a functionalized gold nanoparticle (AuNP) that can serve as a universal carrier for the delivery of DNA oligonucleotides (oligos) into the nucleus. We designed various types of DNA oligos to redirect alternative splicing of pre-mRNAs, such as MCL-1 and BCL-6, and to sequester transcriptional factors, including estrogen receptor α and p53. We successfully delivered the oligos into the nucleus, resulting in the targeted effects. In addition, injection of the antisense DNAs into a xenograft tumor in a mouse model system resulted in inhibited development of the tumor by redirecting the alternative splicing of the pre-mRNA. Our findings show that these nanoconjugates efficiently load and deliver antisense DNAs to redirect gene splicing or double-stranded DNAs to decoy gene transcription by transcriptional factors into mammalian cells and in vivo animals. Therefore, our lego-like AuNP gene delivery system can be used universally to control different biological processes by modulating nuclear gene expression events in living systems.

FOXL2 interacts with steroidogenic factor-1 (SF-1) and represses SF-1-induced CYP17 transcription in granulosa cells.

Mutations in FOXL2 are responsible for blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) type I, in which affected women exhibit premature ovarian failure. FOXL2-null mice showed defects in granulosa cell development during folliculogenesis. We screened a rat ovarian yeast two-hybrid cDNA library to identify FOXL2-interacting proteins and found steroidogenic factor-1 (SF-1). Here, we show that human FOXL2 and SF-1 proteins interact in human granulosa cells and that FOXL2 negatively regulates the transcriptional activation of a steroidogenic enzyme, CYP17, by SF-1. Furthermore, FOXL2 mutants found in blepharophimosis-ptosis-epicanthus inversus syndrome type I patients lost the ability to repress CYP17 induction mediated by SF-1. Chromatin immunoprecipitation and EMSA results further revealed that FOXL2 inhibited the binding of SF-1 to the CYP17 promoter, whereas the FOXL2 mutants failed to block this interaction. Therefore, this study identifies a novel regulatory role for FOXL2 on a key steroidogenic enzyme and provides a possible mechanism by which mutations in FOXL2 disrupt normal ovarian follicle development.

Decolorization of malachite green by cytochrome c in the mitochondria of the fungus Cunninghamella elegans.

We studied the decolorization of malachite green (MG) by the fungus Cunninghamella elegans. The mitochondrial activity for MG reduction was increased with a simultaneous increase of a 9-kDa protein, called CeCyt. The presence of cytochrome c in CeCyt protein was determined by optical absorbance spectroscopy with an extinction coefficient (E(550-535)) of 19.7+/-6.3 mM(-1) cm(-1) and reduction potential of + 261 mV. When purified CeCyt was added into the mitochondria, the specific activity of CeCyt reached 440 +/- 122 micromol min(-1) mg(-1) protein. The inhibition of MG reduction by stigmatellin, but not by antimycin A, indicated a possible linkage of CeCyt activity to the Qo site of the bc1 complex. The RT-PCR results showed tight regulation of the cecyt gene expression by reactive oxygen species. We suggest that CeCyt acts as a protein reductant for MG under oxidative stress in a stationary or secondary growth stage of this fungus.

Functional implications of the conserved action of regulators of ribonuclease activity.

RNase E (Rne) plays a major role in the decay and processing of numerous RNAs in E. coli, and protein inhibitors of RNase E, RraA and RraB, have recently been discovered. Here, we report that coexpression of RraA or RraB reduces the ribonucleolytic activity in rne-deleted E. coli cells overproducing RNase ES, a Streptomyces coelicolor functional ortholog of RNase E, and consequently rescues these cells from growth arrest. These findings suggest that the regulators of ribonuclease activity have a conserved intrinsic property that effectively acts on an RNase E-like enzyme found in a distantly related bacterial species.

Identification of amino acid residues in the catalytic domain of RNase E essential for survival of Escherichia coli: functional analysis of DNase I subdomain.

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.

Inhibitory effects of RraA and RraB on RNAse E-related enzymes imply conserved functions in the regulated enzymatic cleavage of RNA.

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.

Heterogeneous rRNA molecules encoded by Streptomyces coelicolor M145 genome are all expressed and assembled into ribosomes.

The Streptomyces coelicolor M145 genome harbors six copies of divergent rRNA operons that differ at ~0.2% and ~0.6% of the nucleotide positions in small subunit (SSU) and large subunit (LSU) rRNA genes, respectively. When these rRNA genes are expressed, a single cell may harbor three different kinds of SSU rRNA and five kinds of LSU rRNA. Primer extension analyses revealed that all of the heterogeneous rRNA molecules are expressed and assembled into ribosomes. This finding and the maintenance of the intragenomic variability of rRNA operons imply the existence of functional divergence of rRNA species in this developmentally complex microorganism.