Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNA^Tyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNA^Tyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA^Tyr. The endogenous TyrRS and tRNA^Tyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNA^Tyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.     

A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase

Human tryptophanyl-tRNA synthetase (TrpRS) is secreted into the extracellular region of vascular endothelial cells. The splice variant form (mini TrpRS) functions in vascular endothelial cell apoptosis as an angiostatic cytokine. In contrast, the closely related human tyrosyl-tRNA synthetase (TyrRS) functions as an angiogenic cytokine in its truncated form (mini TyrRS). Here, we determined the crystal structure of human mini TrpRS at a resolution of 2.3 A and compared the structure with those of prokaryotic TrpRS and human mini TyrRS. Deletion of the tRNA anticodon-binding (TAB) domain insertion, consisting of eight residues in the human TrpRS, abolished the enzyme's apoptotic activity for endothelial cells, whereas its translational catalysis and cell-binding activities remained unchanged. Thus, we have identified the inserted peptide motif that activates the angiostatic signaling.     

Dissociating quaternary structure regulates cell-signaling functions of a secreted human tRNA synthetase

Many tRNA synthetases are homodimers that are catalytically inactive as monomers. An example is the 528-amino acid human tyrosyl-tRNA synthetase, which is made up of an N-terminal catalytic unit (TyrRS(Mini)) and a 164-amino acid C-domain. Although native TyrRS has no known cytokine functions, natural proteolysis of secreted TyrRS releases TyrRS(Mini), which not only has the same aminoacylation activity as native TyrRS but also has strong activity for stimulating migration of polymorphonuclear leukocytes. The migration-stimulating activity is dependent on an ELR tripeptide motif, similar to that in CXC cytokines like IL-8, and also has the familiar bell-shaped concentration dependence seen for CXC cytokines. Here we show that in contrast to IL-8, where the bell-shaped dependence arises from the effects of CXCR1/2 receptor internalization, TyrRS(Mini) does not induce internalization of CXCR1/2. A rationally designed non-associating monomer and a non-dissociating dimer were constructed. With these constructs, the bell-shaped concentration dependence of leukocyte migration was shown to arise from the agonist (for migration) activity of the catalytically inactive monomer and the antagonist activity of the catalytically active dimer. Thus, the dissociating quaternary structure of TyrRS(Mini) regulates two opposing cytokine activities and suggests the possibility of dissociating quaternary structures regulating novel functions of other tRNA synthetases.     

Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms

The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNA^Tyrs. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNA^Tyr(GΨA). Structural information for TyrRS–tRNA^Tyr complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs–tRNA^Tyrs pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNA^Tyr recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNA^Tyr pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNA^Tyr. On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.     

tRNA-controlled nuclear import of a human tRNA synthetase

Aminoacyl-tRNA synthetases, essential components of the cytoplasmic translation apparatus, also have nuclear functions that continue to be elucidated. However, little is known about how the distribution between cytoplasmic and nuclear compartments is controlled. Using a combination of methods, here we showed that human tyrosyl-tRNA synthetase (TyrRS) distributes to the nucleus and that the nuclear import of human TyrRS is regulated by its cognate tRNA(Tyr). We identified a hexapeptide motif in the anticodon recognition domain that is critical for nuclear import of the synthetase. Remarkably, this nuclear localization signal (NLS) sequence motif is also important for interacting with tRNA(Tyr). As a consequence, mutational alteration of the hexapeptide simultaneously attenuated aminoacylation and nuclear localization. Because the NLS is sterically blocked when the cognate tRNA is bound to TyrRS, we hypothesized that the nuclear distribution of TyrRS is regulated by tRNA(Tyr). This expectation was confirmed by RNAi knockdown of tRNA(Tyr) expression, which led to robust nuclear import of TyrRS. Further bioinformatics analysis showed that to have nuclear import of TyrRS directly controlled by tRNA(Tyr) in higher organisms, the NLS of lower eukaryotes was abandoned, whereas the new NLS was evolved from an anticodon-binding hexapeptide motif. Thus, higher organisms developed a strategy to make tRNA a regulator of the nuclear trafficking of its cognate synthetase. The design in principle should coordinate nuclear import of a tRNA synthetase with the demands of protein synthesis in the cytoplasm.     

Induction of angiogenesis by a fragment of human tyrosyl-tRNA synthetase

The first step of protein synthesis is catalyzed by aminoacyl-tRNA synthetases. In addition, certain mammalian tRNA synthetases link protein synthesis to cytokine signaling pathways. In particular, human tyrosyl-tRNA synthetase (TyrRS) can be split by proteolysis into two fragments having distinct cytokine activities. One of the TyrRS fragments (mini TyrRS) contains features identical to those in CXC chemokines (like interleukin-8) that also act as angiogenic factors. Here mini TyrRS (but not full-length TyrRS) is shown to stimulate chemotaxis of endothelial cells in vitro and stimulate angiogenesis in each of two in vivo animal models. The angiogenic activity of mini TyrRS can be opposed by anti-angiogenic chemokines like IP-10. Thus, a biological fragment of human tyrosyl-tRNA synthetase links protein synthesis to regulation of angiogenesis.     

Mutational switching of a yeast tRNA synthetase into a mammalian-like synthetase cytokine

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. A link was recently established between protein biosynthesis and cytokine signal transduction. Human tyrosyl-tRNA synthetase can be split into two fragments, each of which has a distinct cytokine function. This activity is specific to the human enzyme. It is absent in the enzymes from lower organisms such as bacteria and yeast. Here, yeast tyrosyl-tRNA synthetase (TyrRS), which lacks cytokine activity, was used as a model to explore how a human tyrosyl-tRNA synthetase during evolution acquired novel functions beyond aminoacylation. We found that a rationally designed mutant yeast TyrRS(ELR) gained cytokine function. The mutant yeast enzyme gained this function without sacrifice of aminoacylation activity. Therefore, relatively simple alteration of a basic structural motif imparts cytokine activity to a tRNA synthetase while preserving its canonical function. Further work established that mutational switching of a yeast protein to a mammalian-like cytokine was specific to this synthetase and not to just any yeast ortholog of a mammalian cytokine.     

Identification of Two APOBEC3F Splice Variants Displaying HIV-1 Antiviral Activity and Contrasting Sensitivity to Vif

Approximately half of all human genes undergo alternative mRNA splicing. This process often yields homologous gene products exhibiting diverse functions. Alternative splicing of APOBEC3G (A3G) and APOBEC3F (A3F), the major host resistance factors targeted by the HIV-1 protein Vif, has not been explored. We investigated the effects of alternative splicing on A3G/A3F gene expression and antiviral activity. Three alternatively spliced A3G mRNAs and two alternatively spliced A3F mRNAs were detected in peripheral blood mononuclear cells in each of 10 uninfected, healthy donors. Expression of these splice variants was altered in different cell subsets and in response to cellular stimulation. Alternatively spliced A3G variants were insensitive to degradation by Vif but displayed no antiviral activity against HIV-1. Conversely, alternative splicing of A3F produced a 37-kDa variant lacking exon 2 (A3FΔ2) that was prominently expressed in macrophages and monocytes and was resistant to Vif-mediated degradation. Alternative splicing also produced a 24-kDa variant of A3F lacking exons 2–4 (A3FΔ2–4) that was highly sensitive to Vif. Both A3FΔ2 and A3FΔ2–4 displayed reduced cytidine deaminase activity and moderate antiviral activity. These alternatively spliced A3F gene products, particularly A3FΔ2, were incorporated into HIV virions, albeit at levels less than wild-type A3F. Thus, alternative splicing of A3F mRNA generates truncated antiviral proteins that differ sharply in their sensitivity to Vif.     

Asymmetric structure and domain binding interfaces of human tyrosyl‐tRNA synthetase studied by molecular dynamics simulations

Human tyrosyl‐tRNA synthetase (HsTyrRS) is composed of two structural modules: N‐terminal catalytic core and an EMAP II‐like C‐terminal domain. The structures of these modules are known, but no crystal structure of the full‐length HsTyrRS is currently available. An all‐atom model of the full‐length HsTyrRS was developed in this work. The structure, dynamics, and domain binding interfaces of HsTyrRS were investigated by extensive molecular dynamics (MD) simulations. Our data suggest that HsTyrRS in solution consists of a number of compact asymmetric conformations, which differ significantly by their rigidity, internal mobility, orientation of C‐terminal modules, and the strength of interdomain binding. Interfaces of domain binding obtained in MD simulations are in perfect agreement with our previous coarse‐grained hierarchical rotations technique simulations. Formation of the hydrogen bonds between R93 residue of the ELR cytokine motif and the residues A340 and E479 in the C‐module was observed. This observation supports the idea that the lack of cytokine activity in the full‐length HsTyrRS is explained by interactions between N‐modules and C‐modules, which block the ELR motif. Copyright © 2013 John Wiley & Sons, Ltd.     

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.     

Tissue-specific alternative splicing separates the catalytic and cell signaling functions of human leucyl-tRNA synthetase

The aminoacyl-tRNA synthetases are an ancient and ubiquitous component of all life. Many eukaryotic synthetases balance their essential function, preparing aminoacyl-tRNA for use in mRNA translation, with diverse roles in cell signaling. Herein, we use long-read sequencing to discover a leukocyte-specific exon skipping event in human leucyl-tRNA synthetase (LARS). We show that this highly expressed splice variant, LSV3, is regulated by serine-arginine-rich splicing factor 1 (SRSF1) in a cell-type-specific manner. LSV3 has a 71 amino acid deletion in the catalytic domain and lacks any tRNA leucylation activity in vitro. However, we demonstrate that this LARS splice variant retains its role as a leucine sensor and signal transducer for the proliferation-promoting mTOR kinase. This is despite the exon deletion in LSV3 including a portion of the previously mapped Vps34-binding domain used for one of two distinct pathways from LARS to mTOR. In conclusion, alternative splicing of LARS has separated the ancient catalytic activity of this housekeeping enzyme from its more recent evolutionary role in cell signaling, providing an opportunity for functional specificity in human immune cells.     

The mRNA of Human Cytoplasmic Arginyl-tRNA Synthetase Recruits Prokaryotic Ribosomes Independently

There are two isoforms of cytoplasmic arginyl-tRNA synthetase (hcArgRS) in human cells. The long form is a component of the multiple aminoacyl-tRNA synthetase complex, and the other is an N-terminal truncated form (ΔNhcArgRS), free in the cytoplasm. It has been shown that the two forms of ArgRS arise from alternative translational initiation in a single mRNA. The short form is produced from the initiation at a downstream, in-frame AUG start codon. Interestingly, our data suggest that the alternative translational initiation of hcArgRS mRNA also takes place in Escherichia coli transformants. When the gene encoding full-length hcArgRS was overexpressed in E. coli, two forms of hcArgRS were observed. The N-terminal sequencing experiment identified that the short form was identical to the ΔNhcArgRS in human cytoplasm. By constructing a bicistronic system, our data support that the mRNA encoding the N-terminal extension of hcArgRS has the capacity of independently recruiting E. coli ribosomes. Furthermore, two critical elements for recruiting prokaryotic ribosomes were identified, the “AGGA” core of the Shine-Dalgarno sequence and the “A-rich” sequence located just proximal to the alternative in-frame initiation site. Although the mechanisms of prokaryotic and eukaryotic translational initiation are distinct, they share some common features. The ability of the hcArgRS mRNA to recruit the prokaryotic ribosome may provide clues for shedding light on the mechanism of alternative translational initiation of hcArgRS mRNA in eukaryotic cells.     

Misacylation of yeast amber suppressor tRNA(Tyr) by E. coli lysyl-tRNA synthetase and its effective repression by genetic engineering of the tRNA sequence

Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo.     

Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein

Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multisynthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that participates in tRNA trafficking; we show that tRip also functions as an AIMP. We identified three aaRSs, the glutamyl-tRNA synthetase (ERS), glutaminyl-tRNA synthetase (QRS), and methionyl-tRNA synthetase (MRS), which were specifically coimmunoprecipitated with tRip in Plasmodium berghei blood stage parasites. All four proteins contain an N-terminal glutathione-S-transferase (GST)–like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip–ERS–QRS) and the M-complex (tRip–ERS–MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together, our results demonstrate that neither the singular homodimerization of tRip nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host–pathogen interaction is discussed.     

Tyrosyl-tRNA synthetase from wheat germ

Tyrosyl-tRNA synthetase (TyrRS) was purified 5,000-fold from wheat germ extract by ultracentrifugation, precipitation with ammonium acetate, and column chromatography. Under denaturing conditions the enzyme ran as a single band on SDS-polyacrylamide electrophoresis with an apparent Mr of 55,000. The native molecular weight determined by gel filtration was 110,000, suggesting a quaternary structure of an alpha 2 type for native TyrRS. Purified enzyme activity, based on the aminoacylation reaction, was studied in terms of Mg2+, ATP, pH, and KCl dependence. Optimum concentrations were 6 mM Mg2+, 4 mM ATP, and 200 mM KCl at pH 8. The Km values for ATP, tyrosine, and tRNA were 40, 3.3, and 1.5 microM, respectively. The instability of the TyrRS activity and the methods used for stabilizing it are discussed. In wheat germ extract we found a second tyrosylating activity that works with Escherichia coli tRNA, but not with wheat germ tRNA. We believe that this enzyme is the mitochondrial tyrosyl-tRNA synthetase of wheat germ.     

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

Discrimination between cognate and non-cognate tRNAs by aminoacyl-tRNA synthetases occurs at several steps of the aminoacylation pathway. We have measured changes of solvation and counter-ion distribution at various steps of the aminoacylation pathway of glutamyl- and glutaminyl-tRNA synthetases. The decrease in the association constant with increasing KCl concentration is relatively small for cognate tRNA binding when compared to known DNA–protein interactions. The electro-neutral nature of the tRNA binding domain may be largely responsible for this low ion release stoichiometry, suggesting that a relatively large electrostatic component of the DNA–protein interaction free energy may have evolved for other purposes, such as, target search. Little change in solvation upon tRNA binding is seen. Non-cognate tRNA binding actually increases with increasing KCl concentration indicating that charge repulsion may be a significant component of binding free energy. Thus, electrostatic interactions may have been used to discriminate between cognate and non-cognate tRNAs in the binding step. The catalytic constant of glutaminyl-tRNA synthetase increases with increasing osmotic pressure indicating a water release of 8.4 ± 1.4 mol/mol in the transition state, whereas little change is seen in the case of glutamyl-tRNA synthetase. We propose that the significant amount of water release in the transition state, in the case of glutaminyl-tRNA synthetase, is due to additional contact of the protein with the tRNA in the transition state.     

Escherichia coli tyrosyl- and methionyl-tRNA synthetases display sequence similarity at the binding site for the 3'-end of tRNA

Covalent modification of Escherichia coli tyrosyl-tRNA synthetase (TyrRS) by the 2',3'-dialdehyde derivative of tRNATyr (tRNAox) resulted in a time-dependent inactivation of both ATP-PPi exchange and tRNA aminoacylation activities of the enzyme. In parallel with the inactivation, covalent incorporation of approximately 1 mol of [14C]tRNATyrox/mol of the dimeric synthetase occurred. Intact tRNATyr protected the enzyme against inactivation by the tRNA dialdehyde. Treatment of the TyrRS-[14C]tRNATyr covalent complex with alpha-chymotrypsin produced two labeled peptides (A and B) that were isolated and identified by sequence analysis. Peptides A and B are adjacent and together span residues 227-244 in the primary structure of the enzyme. The three lysine residues in this sequence (lysines-229, -234, and -237) are labeled in a mutually exclusive fashion, with lysine-234 being the most reactive. By analogy with the known three-dimensional structure of the homologous tyrosyl-tRNA synthetase from Bacillus stearothermophilus, these lysines should be part of the C-terminal domain which is presumed to bind the cognate tRNA. Interestingly, the labeled TyrRS structure showed significant similarities to the structure around the lysine residue of E. coli methionyl-tRNA synthetase which is the most reactive toward tRNAMetf(ox) (lysine-335) [Hountondji, C., Blanquet, S., & Lederer, F. (1985) Biochemistry 24, 1175-1180].     

Dominant Intermediate Charcot-Marie-Tooth disorder is not due to a catalytic defect in tyrosyl-tRNA synthetase

Charcot-Marie-Tooth disorder (CMT) is the most common inherited peripheral neuropathy, afflicting 1 in every 2500 Americans. One form of this disease, Dominant Intermediate Charcot-Marie-Tooth disorder type C (DI-CMTC), is due to mutation of the gene encoding the cytoplasmic tyrosyl-tRNA synthetase (TyrRS). Three different TyrRS variants have been found to give rise to DI-CMTC: replacing glycine at position 41 by arginine (G41R), replacing glutamic acid at position 196 by lysine (E196K), and deleting amino acids 153-156 (Δ(153-156)). To test the hypothesis that DI-CMTC is due to a defect in the ability of tyrosyl-tRNA synthetase to catalyze the aminoacylation of tRNA(Tyr), we have expressed each of these variants as recombinant proteins and used single turnover kinetics to characterize their abilities to catalyze the activation of tyrosine and its subsequent transfer to the 3' end of tRNA(Tyr). Two of the variants, G41R and Δ(153-156), display a substantial decrease in their ability to bind tyrosine (>100-fold). In contrast, the E196K substitution does not significantly affect the kinetics for formation of the tyrosyl-adenylate intermediate and actually increases the rate at which the tyrosyl moiety is transferred to tRNA(Tyr). The observation that the E196K substitution does not decrease the rate of catalysis indicates that DI-CMTC is not due to a catalytic defect in tyrosyl-tRNA synthetase.     

RNase P: role of distinct protein cofactors in tRNA substrate recognition and RNA-based catalysis

The Escherichia coli ribonuclease P (RNase P) has a protein component, termed C5, which acts as a cofactor for the catalytic M1 RNA subunit that processes the 5′ leader sequence of precursor tRNA. Rpp29, a conserved protein subunit of human RNase P, can substitute for C5 protein in reconstitution assays of M1 RNA activity. To better understand the role of the former protein, we compare the mode of action of Rpp29 to that of the C5 protein in activation of M1 RNA. Enzyme kinetic analyses reveal that complexes of M1 RNA–Rpp29 and M1 RNA–C5 exhibit comparable binding affinities to precursor tRNA but different catalytic efficiencies. High concentrations of substrate impede the activity of the former complex. Rpp29 itself exhibits high affinity in substrate binding, which seems to reduce the catalytic efficiency of the reconstituted ribonucleoprotein. Rpp29 has a conserved C-terminal domain with an Sm-like fold that mediates interaction with M1 RNA and precursor tRNA and can activate M1 RNA. The results suggest that distinct protein folds in two unrelated protein cofactors can facilitate transition from RNA- to ribonucleoprotein-based catalysis by RNase P.     

Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

The endoribonuclease RNase E of Escherichia coli is an essential enzyme that plays a major role in all aspects of RNA metabolism. In contrast, its paralog, RNase G, seems to have more limited functions. It is involved in the maturation of the 5′ terminus of 16S rRNA, the processing of a few tRNAs, and the initiation of decay of a limited number of mRNAs but is not required for cell viability and cannot substitute for RNase E under normal physiological conditions. Here we show that neither the native nor N-terminal extended form of RNase G can restore the growth defect associated with either the rne-1 or rneΔ1018 alleles even when expressed at very high protein levels. In contrast, two distinct spontaneously derived single amino acid substitutions within the predicted RNase H domain of RNase G, generating the rng-219 and rng-248 alleles, result in complementation of the growth defect associated with various RNase E mutants, suggesting that this region of the two proteins may help distinguish their in vivo biological activities. Analysis of rneΔ1018/rng-219 and rneΔ1018/rng-248 double mutants has provided interesting insights into the distinct roles of RNase E and RNase G in mRNA decay and tRNA processing.