Different cleavage specificities of RNases III from Rhodobacter capsulatus and Escherichia coli.

23S rRNA in Rhodobacter capsulatus shows endoribonuclease III (RNase III)-dependent fragmentation in vivo at a unique extra stem-loop extending from position 1271 to 1331. RNase III is a double strand (ds)-specific endoribonuclease. This substrate preference is mediated by a double-stranded RNA binding domain (dsRBD) within the protein. Although a certain degree of double strandedness is a prerequisite, the question arises what structural features exactly make this extra stem-loop an RNase III cleavage site, distinguishing it from the plethora of stem-loops in 23S rRNA? We used RNase III purified from R.capsulatus and Escherichia coli, respectively, together with well known substrates for E.coli RNase III and RNA substrates derived from the special cleavage site in R.capsulatus 23S rRNA to study the interaction between the Rhodobacter enzyme and the fragmentation site. Although both enzymes are very similar in their amino acid sequence, they exhibit significant differences in binding and cleavage of these in vitro substrates.     

The CafA protein required for the 5'-maturation of 16 S rRNA is a 5'-end-dependent ribonuclease that has context-dependent broad sequence specificity

The CafA protein, which was initially described as having a role in either Escherichia coli cell division or chromosomal segregation, has recently been shown to be required for the maturation of the 5'-end of 16 S rRNA. The sequence of CafA is similar to that of the N-terminal ribonucleolytic half of RNase E, an essential E. coli enzyme that has a central role in the processing of rRNA and the decay of mRNA and RNAI, the antisense regulator of ColE1-type plasmids. We show here that a highly purified preparation of CafA is sufficient in vitro for RNA cutting. We detected CafA cleavage of RNAI and a structured region from the 5'-untranslated region of ompA mRNA within segments cleavable by RNaseE, but not CafA cleavage of 9 S RNA at its "a" RNase E site. The latter is consistent with the finding that the generation of 5 S rRNA from its 9 S precursor can be blocked by inactivation of RNase E in cells that are wild type for CafA. Interestingly, however, a decanucleotide corresponding in sequence to the a site of 9 S RNA was cut efficiently indicating that cleavage by CafA is regulated by the context of sites within structured RNAs. Consistent with this notion is our finding that although 23 S rRNA is stable in vivo, a segment from this RNA is cut efficient by CafA at multiple sites in vitro. We also show that, like RNase E cleavage, the efficiency of cleavage by CafA is dependent on the presence of a monophosphate group on the 5'-end of the RNA. This finding raises the possibility that the context dependence of cleavage by CafA may be due at least in part to the separation of a cleavable sequence from the 5'-end of an RNA. Comparison of the sites surrounding points of CafA cleavage suggests that this enzyme has broad sequence specificity. Together with the knowledge that CafA can cut RNAI and ompA mRNA in vitro within segments whose cleavage in vivo initiates the decay of these RNAs, this finding suggests that CafA may contribute at some point during the decay of many RNAs in E. coli.     

Kinetic analysis of precursor M1 RNA molecules for exploring substrate specificity of the N-terminal catalytic half of RNase E

To gain insight into the mechanism by which the sequence at the rne-dependent site of substrate RNA affects the substrate specificity of Escherichia coli RNase E, we performed kinetic analysis of the cleavage of precursor M1 RNA molecules containing various sequences at the rne-dependent site by the N-terminal catalytic half of RNase E (NTH-RNase E). NTH-RNase E displayed higher K(m) and k(cat) values for more specific substrates. The retention of single strandedness at the rne-dependent site was essential for cleavage efficiency. Moreover, the loss of single-strandedness was accompanied by a decrease in both the K(m) and k(cat) values.     

Determination of the catalytic parameters of the N-terminal half of Escherichia coli ribonuclease E and the identification of critical functional groups in RNA substrates

Ribonuclease E is required for the rapid decay and correct processing of RNA in Escherichia coli. A detailed understanding of the hydrolysis of RNA by this and related enzymes will require the integration of structural and molecular data with quantitative measurements of RNA hydrolysis. Therefore, an assay for RNaseE that can be set up to have relatively high throughput while being sensitive and quantitative will be advantageous. Here we describe such an assay, which is based on the automated high pressure liquid chromatography analysis of fluorescently labeled RNA samples. We have used this assay to optimize reaction conditions, to determine for the first time the catalytic parameters for a polypeptide of RNaseE, and to investigate the RNaseE-catalyzed reaction through the modification of functional groups within an RNA substrate. We find that catalysis is dependent on both protonated and unprotonated functional groups and that the recognition of a guanosine sequence determinant that is upstream of the scissile bond appears to consist of interactions with the exocyclic 2-amino group, the 7N of the nucleobase and the imino proton or 6-keto group. Additionally, we find that a ribose-like sugar conformation is preferred in the 5'-nucleotide of the scissile phosphodiester bond and that a 2'-hydroxyl group proton is not essential. Steric bulk at the 2' position in the 5'-nucleotide appears to be inhibitory to the reaction. Combined, these observations establish a foundation for the functional interpretation of a three-dimensional structure of the catalytic domain of RNaseE when solved.     

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.     

Antisense RNA based down-regulation of RNaseE in E.coli

Background  

Messenger RNA decay is an important mechanism for controlling gene expression in all organisms. The rate of the mRNA degradation directly affects the steady state concentration of mRNAs and therefore influences the protein synthesis. RNaseE has a key importance for the general mRNA decay in E.coli. While RNaseE initiates the degradation of most mRNAs in E.coli, it is likely that the enzyme is also responsible for the degradation of recombinant RNAs. As RNaseE is essential for cell viability and knockout mutants cannot be cultured, we investigated the possibility for a down-regulation of the intracellular level of RNaseE by antisense RNAs. During this study, an antisense RNA based approach could be established which revealed a strong reduction of the intracellular level of RNaseE in E.coli.     

Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli.

The role of 2'-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate. The presence of the 2'-hydroxyl groups of nucleotides at positions -1 and -2 in the leader sequence and at position 1, as well as at the first C in the 3'-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA. The 2'-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg2+. An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit. Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro. The identity of the nucleotide at position 1 in the substrate, the adjacent Mg(2+)-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.     

Ectopic RNase E sites promote bypass of 5'-end-dependent mRNA decay in Escherichia coli

In Escherichia coli, 5'-terminal stem-loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear. A synthetic 5'-terminal hairpin stabilizes the rpsT mRNA sixfold. This stabilization is dependent on efficient translational initiation and ribosome transit through at least two-thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA. Insertion of a 12-15 residue 'ectopic' RNase E cleavage site from either the rne leader or 9S pre-rRNA into the 5'-non-coding region of the rpsT mRNA significantly reduces the stabilizing effect of the terminal stem-loop, dependent on RNase E. A similar insertion into the rpsT coding sequence is partially destabilizing. These findings demonstrate that RNase E can bypass an interaction with the 5'-terminus, and exploit an alternative 'internal entry' pathway. We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5'-termini, the use of internal entry for bypass of barriers to decay, 'ectopic sites' and the role of translating ribosomes.     

The P3 domain of E. coli ribonuclease P RNA can be truncated and replaced

We prepared some truncated and replaced P3 mutants of Escherichia coli RNase P RNA, and used them to examine the RNase P ribozyme and holoenzyme reactions of a pre-tRNA substrate. The results indicated that mutations in the P3 domain did not affect the cleavage site selection of the pre-tRNA substrate, but did affect the efficiency of cleavage of the substrate. Results of stepwise truncation of the P3 domain and its replacement by the TAR sequence showed that the P3 domain of the E. coli RNase P was able to be truncated to certain length and was replaceable, but could not be deleted in the ribozyme.     

Investigating the structure of human RNase H1 by site-directed mutagenesis

In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.     

Efficient cleavage of pre-tRNAs by E. coli RNAse P RNA requires the 2'-hydroxyl of the ribose at the cleavage site.

RNAse P cleaves pre-tRNAs to liberate 5'-flanks and 5'-matured, 5'-phosphorylated tRNAs. It is not evident if the 2'-hydroxyls of the ribose moieties in the substrate are involved in the reaction. To study their influence in two different pre-tRNAs, we have modified specifically the 2'-hydroxyl groups at the cleavage site and in neighbouring positions. We have shown that these hydroxyls are important but not essential for the processing of these substrates by E. coli RNase P RNA (M1 RNA). The reduction in the catalytic efficiency was moderate for 2'-deoxy and severe for 2'-methoxy substitutions at the cleavage site. Additional effects of modifications in neighbouring positions were smaller. Based on our data we suggest that the modifications do not interfere with binding of the substrate, whereas they prevent an optimal steric arrangement for the hydrolysis reaction.     

Protein cofactor-dependent acquisition of novel catalytic activity by the RNase P ribonucleoprotein of E. coli

Escherichia coli RNase P derivatives were evolved in vitro for DNA cleavage activity. Ribonucleoproteins sampled after ten generations of selection show a >400-fold increase in the first-order rate constant (k(cat)) on a DNA substrate, reflecting a significant improvement in the chemical cleavage step. This increase is offset by a reduction in substrate binding, as measured by K(M). We trace the catalytic enhancement to two ubiquitous A-->U sequence changes at positions 136 and 333 in the M1 RNA component, positions that are phylogenetically conserved in the Eubacteria. Furthermore, although the mutations are located in different folding domains of the catalytic RNA, the first in the substrate binding domain, the second near the catalytic core, their effect on catalytic activity is significantly influenced by the presence of the C5 protein. The activity of the evolved ribonucleoproteins on both pre-4.5 S RNA and on an RNA oligo substrate remain at wild-type levels. In contrast, improved DNA cleavage activity is accompanied by a 500-fold decrease in pre-tRNA cleavage efficiency (k(cat)/K(M)). The presence of the C5 component does not buffer this tradeoff in catalytic activities, despite the in vivo role played by the C5 protein in enhancing the substrate versatility of RNase P. The change at position 136, located in the J11/12 single-stranded region, likely alters the geometry of the pre-tRNA-binding cleft and may provide a functional explanation for the observed tradeoff. These results thus shed light both on structure/function relations in E. coli RNase P and on the crucial role of proteins in enhancing the catalytic repertoire of RNA.     

Fluorescence-based incision assay for human XPF-ERCC1 activity identifies important elements of DNA junction recognition

The structure-specific endonuclease activity of the human XPF-ERCC1 complex is essential for a number of DNA processing mechanisms that help to maintain genomic integrity. XPF-ERCC1 cleaves DNA structures such as stem-loops, bubbles or flaps in one strand of a duplex where there is at least one downstream single strand. Here, we define the minimal substrate requirements for cleavage of stem-loop substrates allowing us to develop a real-time fluorescence-based assay to measure endonuclease activity. Using this assay, we show that changes in the sequence of the duplex upstream of the incision site results in up to 100-fold variation in cleavage rate of a stem-loop substrate by XPF-ERCC1. XPF-ERCC1 has a preference for cleaving the phosphodiester bond positioned on the 3'-side of a T or a U, which is flanked by an upstream T or U suggesting that a T/U pocket may exist within the catalytic domain. In addition to an endonuclease domain and tandem helix-hairpin-helix domains, XPF has a divergent and inactive DEAH helicase-like domain (HLD). We show that deletion of HLD eliminates endonuclease activity and demonstrate that purified recombinant XPF-HLD shows a preference for binding stem-loop structures over single strand or duplex alone, suggesting a role for the HLD in initial structure recognition. Together our data describe features of XPF-ERCC1 and an accepted model substrate that are important for recognition and efficient incision activity.     

Structural and mutational studies of the catalytic domain of colicin E5: a tRNA-specific ribonuclease

Colicin E5 specifically cleaves four tRNAs in Escherichia coli that contain the modified nucleotide queuosine (Q) at the wobble position, thereby preventing protein synthesis and ultimately resulting in cell death. Here, the crystal structure of the catalytic domain of colicin E5 (E5-CRD) from E. coli was determined at 1.5 A resolution. Unexpectedly, E5-CRD adopts a core folding with a four-stranded beta-sheet packed against an alpha-helix, seen in the well-studied ribonuclease T1 despite a lack of sequence similarity. Beyond the core catalytic domain, an N-terminal helix, a C-terminal beta-strand and loop, and an extended internal loop constitute an RNA binding cleft. Mutational analysis identified five amino acids that were important for tRNA substrate binding and cleavage by E5-CRD. The structure, together with the mutational study, allows us to propose a model of colicin E5-tRNA interactions, suggesting the molecular basis of tRNA substrate recognition and the mechanism of tRNA cleavage by colicin E5.     

Acquisition of novel catalytic activity by the M1 RNA ribozyme: the cost of molecular adaptation.

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4. 5S RNA. In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA. This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates. This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding. The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available.     

Evidence for induced fit in bacterial RNase P RNA-mediated cleavage

RNase P with its catalytic RNA subunit is involved in the processing of a number of RNA precursors with different structures. However, precursor tRNAs are the most abundant substrates for RNase P. Available data suggest that a tRNA is folded into its characteristic structure already at the precursor state and that RNase P recognizes this structure. The tRNA D-/T-loop domain (TSL-region) is suggested to interact with the specificity domain of RNase P RNA while residues in the catalytic domain interact with the cleavage site. Here, we have studied the consequences of a productive interaction between the TSL-region and its binding site (TBS) in the specificity domain using tRNA precursors and various hairpin-loop model substrates. The different substrates were analyzed with respect to cleavage site recognition, ground-state binding, cleavage as a function of the concentration of Mg(2+) and the rate of cleavage under conditions where chemistry is suggested to be rate limiting using wild-type Escherichia coli RNase P RNA, M1 RNA, and M1 RNA variants with structural changes in the TBS-region. On the basis of our data, we conclude that a productive TSL/TBS interaction results in a conformational change in the M1 RNA substrate complex that has an effect on catalysis. Moreover, it is likely that this conformational change comprises positioning of chemical groups (and Mg(2+)) at and in the vicinity of the cleavage site. Hence, our findings are consistent with an induced-fit mechanism in RNase P RNA-mediated cleavage.     

Substrate recognition by a eukaryotic RNase III: the double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5'-AGNN-3' tetraloop

Rnt1p is an RNase III homolog from budding yeast, required for processing snRNAs, snoRNAs, and rRNA. Numerous Rnt1p RNA substrates share potential to form a duplex structure with a terminal four-base loop with the sequence AGNN. Using a synthetic RNA modeled after the 25S rRNA 3' ETS cleavage site we find that the AGNN loop is an important determinant of substrate selectivity. When this loop sequence is altered, the rate of Rnt1p cleavage is reduced. The reduction in cleavage rate can be attributed to reduced binding of the mutant substrate as measured by a gel-shift assay. Deletion of the nonconserved N-terminal domain of Rnt1p does not affect cleavage site choice or the ability of the enzyme to distinguish substrates that contain the AGNN loop, indicating that this region is not required for selective cleavage. Strikingly, a recombinant fragment of Rnt1p containing little more than the dsRBD is able to discriminate between wild-type and mutant loop sequences in a binding assay. We propose that a major determinant of AGNN loop recognition by Rnt1p is present in its dsRBD.     

Cleavage efficiencies of model substrates for ribonuclease P from Escherichia coli and Thermus thermophilus.

We compared cleavage efficiencies of mono-molecular and bipartite model RNAs as substrates for RNase P RNAs (M1 RNAs) and holoenzymes from E. coli and Thermus thermophilus, an extreme thermophilic eubacterium. Acceptor stem and T arm of pre-tRNA substrates are essential recognition elements for both enzymes. Impairing coaxial stacking of acceptor and T stems and omitting the T loop led to reduced cleavage efficiencies. Small model substrates were less efficiently cleaved by M1 RNA and RNase P from T. thermophilus than by the corresponding E. coli activities. Competition kinetics and gel retardation studies showed that truncated tRNA substrates are less tightly bound by RNase P and M1 RNA from both bacteria. Our data further indicate that (pre-)tRNA interacts stronger with E. coli than T. thermophilus M1 RNA. Thus, low cleavage efficiencies of truncated model substrates by T. thermophilus RNase P or M1 RNA could be explained by a critical loss of important contact points between enzyme and substrate. In addition, acceptor stem--T arm substrates, composed of two synthetic RNA fragments, have been designed to mimic internal cleavage of any target RNA molecule available for base pairing.