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

Abstract

SummaryThe bacterial tRNA Lys -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.

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

“…GTP and dTTP probably exert their respective ACNase activating and stabilizing functions by interacting with PrrC's N-domains. This is suggested by their binding to full-sized PrrC protein or PrrC's isolated N-domains with vastly differing affinities (mMand µM-range, respectively) and without displacing each other (Amitsur et al, 2003;Blanga-Kanfi et al, 2006;and unpublished data).…”

“…2A). This PrrC Box motif is highly degenerate (or rudimental) in RloC and is missing from other ABC ATPases and any other protein in the public database (Amitsur et al, 2003;Blanga-Kanfi et al, 2006). The PrrC Box candidates as a Y-loop substitute, responsible perhaps for PrrC's unusual specificity, the ability to simultaneously interact with its two different effector nucleotides GTP and dTTP (BlangaKanfi et al, 2006;unpublished data (Gerlt, 1993;Steyaert, 1997), in the PrrC/RloC triad Glu and His could function as respective general base and acid catalysts while Arg could stabilize the pentameric transition state phosphate.…”

“…Thus, in vitro activation of the latent ACNase requires besides the Stp peptide, the DNA tethered to EcoprrI, GTP hydrolysis and the presence of dTTP. In contrast, the overt activity of the core ACNase is refractory to Stp, DNA and GTP but rapidly decays without dTTP (Amitsur et al, 2003;Blanga-Kanfi et al, 2006). These differences have been taken to indicate that Stp triggers the activation of the latent ACNase, GTP hydrolysis drives conformational changes needed to turn it on while the binding of dTTP stabilizes the ACNase once activated.…”

“…The biological significance of PrrC's idiosyncratic interaction with dTTP has been hinted at by the dramatic increase in the cellular level of dTTP early in phage T4 infection, when the ACNase is induced (Amitsur et al, 2003;Blanga-Kanfi et al, 2006). The increased level of dTTP benefits the phage by safeguarding effective and faithful replication of its AT-rich DNA.…”

“…PrrC's dissociation from the latent holoenzyme may be accidental or due to EcoprrI's disruption in response to DNA damage (Restriction Alleviation, RA) (Makovets et al, 2004) (see also section 3.6). Free PrrC's cytotoxicity has been indicated by the coincident inactivation of prrC and linked hsd genes, by the self-limiting expression of free PrrC (Meidler et al, 1999;Blanga-Kanfi et al, 2006) and the rapid in vivo inactivation of the core ACNase (Amitsur et al, 2003). The ACNase enhancing effects of dTTP's accretion during phage T4 infection (Klaiman & Kaufmann, 2011) and in vitro stabilization of the core ACNase by dTTP (Amitsur et al, 2003) suggest that the in vivo instability of the core ACNase owes to the relatively low dTTP level in the uninfected cell.…”