Bacteriophage T4-encoded Stp can be replaced as activator of anticodon nuclease by a normal host cell metabolite

The bacterial tRNALys-specific anticodon nuclease is known as a phage T4 exclusion system. In the uninfected host cell anticodon nuclease is kept latent due to the association of its core protein PrrC with the DNA restriction-modification endonuclease EcoprrI. Stp, the T4-encoded peptide inhibitor of EcoprrI activates the latent enzyme. Previous in vitro work indicated that the activation by Stp is sensitive to DNase and requires added nucleotides. Biochemical and mutational data reported here suggest that Stp activates the latent holoenzyme when its EcoprrI component is tethered to a cognate DNA substrate. Moreover, the activation is driven by GTP hydrolysis, possibly mediated by the NTPase domain of PrrC. The data also reveal that Stp can be replaced as the activator of latent anticodon nuclease by certain pyrimidine nucleotides, the most potent of which is dTTP. The activation by dTTP likewise requires an EcoprrI DNA substrate and GTP hydrolysis but involves a different form of the latent holoenzyme/DNA complex. Moreover, whereas Stp relays its activating effect through EcoprrI, dTTP targets PrrC. The activation of the latent enzyme by a normal cell constituent hints that anticodon nuclease plays additional roles, other than warding off phage T4 infection.     

Parallel dimerization of a PrrC-anticodon nuclease region implicated in tRNALys recognition

The optional Escherichia coli restriction tRNase PrrC represents a family of potential antiviral devices widespread among bacteria. PrrC comprises a functional C-domain of unknown structure and regulatory ABC/ATPase-like N-domain. The possible involvement of a C-domain sequence in tRNA^Lys recognition was investigated using a matching end-protected 11-meric peptide. This mimic, termed here LARP (Lys-anticodon recognizing peptide) UV-cross-linked tRNA^Lys anticodon stem-loop (ASL) analogs and inhibited their PrrC-catalyzed cleavage. Trimming LARP or introducing in it inactivating PrrC missense mutations impaired these activities. LARP appeared to mimic its matching protein sequence in ability to dimerize in parallel, as inferred from the following results. First, tethering Cys to the amino- or carboxy-end of LARP dramatically enhanced the ASL-cross-linking and PrrC-inhibiting activities under suitable redox conditions. Second, Cys-substitutions in a C-domain region containing the sequence corresponding to LARP elicited specific intersubunit cross-links. The parallel dimerization of PrrC's C-domains and expected head-to-tail dimerization of its N-domains further suggest that the NTPase and tRNA^Lys-binding sites of PrrC arise during distinct assembly stages of its dimer of dimers form.     

Determinants of eukaryal cell killing by the bacterial ribotoxin PrrC

tRNA damage inflicted by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies an antiviral response to phage T4 infection. PrrC homologs are present in many bacterial proteomes, though their biological activities are uncharted. PrrCs consist of two domains: an N-terminal NTPase module related to the ABC family and a distinctive C-terminal ribonuclease module. In this article, we report that the expression of EcoPrrC in budding yeast is fungicidal, signifying that PrrC is toxic in a eukaryon in the absence of other bacterial or viral proteins. Whereas Streptococcus PrrC is also toxic in yeast, Neisseria and Xanthomonas PrrCs are not. Via analysis of the effects of 118 mutations on EcoPrrC toxicity in yeast, we identified 22 essential residues in the NTPase domain and 11 in the nuclease domain. Overexpressing PrrCs with mutations in the NTPase active site ameliorated the toxicity of wild-type EcoPrrC. Our findings support a model in which EcoPrrC toxicity is contingent on head-to-tail dimerization of the NTPase domains to form two composite NTP phosphohydrolase sites. Comparisons of EcoPrrC activity in a variety of yeast genetic backgrounds, and the rescuing effects of tRNA overexpression, implicate tRNA^Lys(UUU) as a target of EcoPrrC toxicity in yeast.     

Detection of anticodon nuclease residues involved in tRNALys cleavage specificity

The tRNALys-specific anticodon nuclease exists in latent form in Escherichia coli strains containing the optional prr locus. The latency is a result of a masking interaction between the anticodon nuclease core-polypeptide PrrC and the Type IC DNA restriction-modification enzyme EcoprrI. Activation of the latent enzyme by phage T4-infection elicits cleavage of tRNALys 5' to the wobble base, yielding 5'-OH and 2', 3'-cyclic phosphate termini. The N-proximal half of PrrC has been implicated with (A/G) TPase and EcoprrI interfacing activities. Therefore, residues involved in recognition and cleavage of tRNALys were searched for at the C-half. Random mutagenesis of the low-G+C portion encoding PrrC residues 200-313 was performed, followed by selection for loss of anticodon nuclease-dependent lethality and production of full-sized PrrC-like protein. This process yielded a cluster of missense mutations mapping to a region highly conserved between PrrC and two putative Neisseria meningitidis MC58 homologues. This cluster included two adjacent members that relaxed the inherent enzyme's cleavage specificity. We also describe another mode of relaxed specificity, due to mere overexpression of PrrC. This mode was shared by wild-type PrrC and the other mutant alleles. The additional substrates recognised under the promiscuous conditions had, in general, anticodons resembling that of tRNALys. Taken together, the data suggest that the anticodon of tRNALys harbours anticodon nuclease identity elements and implicates a conserved region in PrrC in their recognition.     

A DNA break inducer activates the anticodon nuclease RloC and the adaptive immunity in Acinetobacter baylyi ADP1

Double-stranded DNA breaks (DSB) cause bacteria to augment expression of DNA repair and various stress response proteins. A puzzling exception educes the anticodon nuclease (ACNase) RloC, which resembles the DSB responder Rad50 and the antiviral, translation-disabling ACNase PrrC. While PrrC''s ACNase is regulated by a DNA restriction-modification (R-M) protein and a phage anti-DNA restriction peptide, RloC has an internal ACNase switch comprising a putative DSB sensor and coupled ATPase. Further exploration of RloC''s controls revealed, first, that its ACNase is stabilized by the activating DNA and hydrolysed nucleotide. Second, DSB inducers activated RloC''s ACNase in heterologous contexts as well as in a natural host, even when R-M deficient. Third, the DSB-induced activation of the indigenous RloC led to partial and temporary disruption of tRNA^Glu and tRNA^Gln. Lastly, accumulation of CRISPR-derived RNA that occurred in parallel raises the possibility that the adaptive immunity and RloC provide the genotoxicated host with complementary protection from impending infections.     

Structural relationship between tRNA2 Lys and tRNA4 Lys from mouse lymphoma cells

Summary Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNA₂ ^Lys and tRNA₄ ^Lys, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNA₄ ^Lys has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t⁶A^*, replacing t⁶A. This unidentified nucleotide is not a hypomodified form of t⁶A. Thus, tRNA4ys is not a simple precursor of tRNA₂ ^Lys. Both tRNAs have an unidentified nucleotide, U^**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNA₂ ^Lys and tRNA₄ ^Lys were obtained. The distinctions between tRNA₂ ^Lys and tRNA₄ ^Lys may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5,5-P₁,P₄-tetraphosphate, a putative signal in DNA replication.     

Phage T4‐induced DNA breaks activate a tRNA repair‐defying anticodon nuclease

The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4‐excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction‐modification (RM) protein and turned on by a phage‐encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double‐stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi RloC expressed in Escherichia coli is activated by wild‐type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction.     

tRNA2Lys gene clusters in Drosophila

We have isolated segments of Drosophila melanogaster DNA that contain two clusters of tRNA₂^Lys genes. In one segment, pPW511, there is a cluster of three of these genes surrounded by other tRNA genes. Two other segments, pPW516 and pPW541. share a 3 × 10³ base-pair region that has a cluster of four tRNA₂^Lys genes. This cluster is flanked by 20 × 10³ base-pairs of DNA that does not appear to have other tRNA genes. The tRNA genes in both clusters are irregularly spaced and are intermingled with moderately repetitive DNA. Each cluster is present once or perhaps twice in the haploid genome and has the same arrangement of restriction endonuclease sites in the genomic DNA as in the isolated, cloned DNA. In situ hybridization to polytene chromosomes localized the pPW511 cluster to the 42A region and the pPW516/541 cluster to the 42E region. Another region, 50B, also contains tRNA₂^Lys genes. In sum, these cloned tRNA₂^Lys genes account for most of this gene family and are irregularly spaced in two clusters.     

Cleavage of the HIV replication primer tRNALys,3 in human cells expressing bacterial anticodon nuclease.

Anticodon nuclease is a bacterial restriction enzyme directed against tRNA(Lys). We report that anticodon nuclease also cleaves mammalian tRNA(Lys) molecules, with preference and site specificity shown towards the natural substrate. Expression of the anticodon nuclease core polypeptide PrrC in HeLa cells from a recombinant vaccinia virus elicited cleavage of intracellular tRNA(Lys),3. The data justify an inquiry into the possible application of anticodon nuclease as an inhibitor of tRNA(Lys),3-primed HIV replication. They also indicate that the anticodon region of tRNA(Lys) is a substrate recognition site and suggest that PrrC harbors the enzymatic activity.     

Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an ‘uncloneable’ phenotype

Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors. For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome. All of 50 recombinant M13 clones containing this ‘uncloneable’ fragment had one or more changes in nucleotide sequence. Each clone contained at least one alteration in two nucleotide positions within the tRNA^Thr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNA^Thr. These results imply that the HeLa mitochondrial tRNA^Thr gene is responsible for the ‘uncloneable’ phenotype of this region of human mitochondrial (mt) DNA.A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNA^Thr gene. 56 mutations were single-base substitutions; 5 were deletions. Approximately 80% of the base substitution mutations were A:T → G:C transitions. A preference for A:T → G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals. This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin.     

Codon:Anticodon and anticodon:Anticodon interaction. Evaluation of equilibrium and kinetic parameters of complexes involving a G:U wobble

In order to learn about the effect of the G:U wobble interaction we characterized the codon:anticodon binding between triplets: UUC, UUU and yeast tRNA^Phe (anticodon GmAA) as well as the anticodon:anticodon binding between Escherichia coli tRNA^Glu₂, E. coli tRNA^Lys (anticodons: mam⁵s²UUC, and mam⁵s²UUU, respectively) and tRNA^Phe from yeast and E. coli (anticodon GAA) using equilibrium fluorescence titrations and temperature jump measurements with fluorescence and absorption detection. The difference in stability constants between complexes involving a G:U pair rather than a usual G:C basepair is in the range of one order of magnitude and is mainly due to the shorter lifetime of the complex involving G:U in the wobble position. This difference is more pronounced when the codon triplet is structured, i.e., is built in the anticodon loop of a tRNA. The reaction enthalpies of the anticodon:anticodon complexes involving G:U mismatching were found to be about 4 kcal/mol smaller, and the melting temperatures more than 20°C lower, than those of the corresponding complexes with the G:C basepair. The results are discussed in terms of different strategies that might be used in the cell in order to minimize the effect of different lifetimes of codon-tRNA complexes. Differences in these lifetimes may be used for the modulation of the translation efficiency.     

RloC: a wobble nucleotide-excising and zinc-responsive bacterial tRNase

The conserved bacterial protein RloC, a distant homologue of the tRNA^Lys anticodon nuclease (ACNase) PrrC, is shown here to act as a wobble nucleotide-excising and Zn⁺⁺-responsive tRNase. The more familiar PrrC is silenced by a genetically linked type I DNA restriction-modification (R-M) enzyme, activated by a phage anti-DNA restriction factor and counteracted by phage tRNA repair enzymes. RloC shares PrrC's ABC ATPase motifs and catalytic ACNase triad but features a distinct zinc-hook/coiled-coil insert that renders its ATPase domain similar to Rad50 and related DNA repair proteins. Geobacillus kaustophilus RloC expressed in Escherichia coli exhibited ACNase activity that differed from PrrC's in substrate preference and ability to excise the wobble nucleotide. The latter specificity could impede reversal by phage tRNA repair enzymes and account perhaps for RloC's more frequent occurrence. Mutagenesis and functional assays confirmed RloC's catalytic triad assignment and implicated its zinc hook in regulating the ACNase function. Unlike PrrC, RloC is rarely linked to a type I R-M system but other genomic attributes suggest their possible interaction in trans . As DNA damage alleviates type I DNA restriction, we further propose that these related perturbations prompt RloC to disable translation and thus ward off phage escaping DNA restriction during the recovery from DNA damage.     

Loss of Anticodon Wobble Uridine Modifications Affects tRNALys Function and Protein Levels in Saccharomyces cerevisiae

In eukaryotes, wobble uridines in the anticodons of tRNA^Lys _UUU, tRNA^Glu _UUC and tRNA^Gln _UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm⁵s²U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm⁵ side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm⁵s²U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm⁵U and s²U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm⁵s²U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA^Lys _UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm⁵ and s² wobble uridine modification that could account for growth impairment and mainly originates from tRNA^Lys _UUU hypomodification and malfunction.     

Construction,aminoacylation and 80 S ribosomal complex formation with a yeast initiator tRNA having an arginine CCU anticodon

The digestion of yeast initiator methionine tRNA with mung bean nuclease and U₂ ribonuclease yielded 5'- and 3'-fragments, respectively. These two fragments together represent the entire tRNA sequence except for A₃₅, the central nucleotide of the anticodon, and the CCA terminus. Using RNA ligase, a cytosine was added and the anticodon loop having a C₃₅ was reformed. Subsequent treatment of this product with CCA-transferase yielded a full-length methionine tRNA having an arginine CCU anticodon. This recombinant tRNA^Met (CCU) was charged with methionine by the yeast tRNA synthetase. Aminoacylation of the recombinant was however less extensive than in the case of native tRNA^Met (CAU). After aminoacylation the recombinant tRNA formed an 80 S ribosomal complex.     

HSD restriction-modification proteins partake in latent anticodon nuclease.

Phage T4-induced anticodon nuclease triggers cleavage-ligation of the host tRNA(Lys). The enzyme is encoded in latent form by the optional Escherichia coli locus prr and is activated by the product of the phage stp gene. Anticodon nuclease latency is attributed to the masking of the core function prrC by flanking elements homologous with type I restriction-modification genes (prrA-hsdM and prrD-hsdR). Activation of anticodon nuclease in extracts of uninfected prr+ cells required synthetic Stp, ATP and GTP and appeared to depend on endogenous DNA. Stp could be substituted by a small, heat-stable E. coli factor, hinting that anticodon nuclease may be mobilized in cellular situations other than T4 infection. Hsd antibodies recognized the anticodon nuclease holoenzyme but not the prrC-encoded core. Taken together, these data indicate that Hsd proteins partake in the latent ACNase complex where they mask the core factor PrrC. Presumably, this masking interaction is disrupted by Stp in conjunction with Hsd ligands. The Hsd-PrrC interaction may signify coupling and mutual enhancement of two prokaryotic restriction systems operating at the DNA and tRNA levels.