HSD restriction-modification proteins partake in latent anticodon nuclease.

Abstract

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.

Structural Features of tRNALys Favored by Anticodon Nuclease as Inferred from Reactivities of Anticodon Stem and Loop Substrate Analogs

“…3 Additional protection against nonspecific degradation was afforded by including in the reaction mixture carrier poly(U), increasing the trimethylamine-N-oxide concentration to 1.5 M and lowering the reaction temperature from 10 to 0°C. The standard reaction mixtures (10 l) contained 5 l of the partially purified core ACNase (D222E allele, Superdex 200 peak activity fraction of ϳ270 kDa) containing 6 ng of PrrC, 36 mM NH 4 Cl, 6 mM Tris-HCl buffer, pH 7.5, 9 mM MgCl 2 , 3 mM 2-mercaptoethanol, 50 -100 ng of poly(U), 1.5 M trimethylamine-N-oxide, 6% glycerol, protease inhibitor mixture (Complete, Mini, Roche Molecular Biochemicals, diluted according to manufacturers instructions), 2-3 fmol of 5Ј-32 P-labeled ASL or E. coli tRNA Lys labeled with 32 P at the ACNase cleavage junction. The products were deproteinized and separated by denaturing polyacrylamide-urea gel electrophoresis.…”

“…It comprises the core ACNase polypeptide PrrC and type IC DNA restriction-modification enzyme EcoprrI that masks PrrCs activity (2)(3)(4)(5). The phage T4-coded peptide Stp inhibits EcoprrI DNA restriction and activates ACNase (6).…”

“…Interestingly, among the PrrC D287 replacement mutants expressed in mammalian cells D287Q showed the highest activity with tRNA , cleaving it to a severalfold higher extent than wild type PrrC. 4 The efficient cleavage of the isolated ASL domain by ACNase also suggests that partially melted tRNA annealed to the HIV-1 genomic RNA will be recognized by ACNase since the native ASL conformation would be retained in the primer-template complex (27,28). This expectation has been recently confirmed.…”

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair

“…The normally latent EcoPrrC nuclease is switched on by the virus-encoded Stp peptide synthesized early during T4 infection (Amitsur et al 1989(Amitsur et al , 1992Penner et al 1995). The activated form of EcoPrrC incises the tRNA Lys(UUU) anticodon loop at a single site 59 of the wobble uridine, leaving 29,39 cyclic phosphate and 59-OH ends at the break.…”

RloC: A Translation-Disabling tRNase Implicated in Phage Exclusion During Recovery from DNA Damage

“…These facts reinforced the notion that Pnk and Rnl1 cooperate in RNA nick repair. They also led to the detection of the prr-encoded latent ACNase comprising the core ACNase PrrC and PrrC's silencing partner, the associated type Ic DNA restriction-modification (R-M) system EcoprrI (Levitz et al, 1990;Linder et al, 1990;Amitsur et al, 1992;Tyndall et al, 1994). EcoprrI and PrrC are also genetically linked, the ACNase core gene prrC is flanked by the genes encoding the three R-M subunit types hsdMSR/prrABD (Fig.…”