RtcB is an atypical RNA ligase that joins either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. In contrast to typical RNA ligases, which rely on ATP and Mg(II), catalysis by RtcB is dependent on GTP and Mn(II) with ligation proceeding through a covalent RtcB–histidine–GMP intermediate. Here, we present three structures of Pyrococcus horikoshii RtcB complexes that capture snapshots along the entire guanylylation pathway. These structures show that prior to binding GTP, a single manganese ion (Mn1) is bound to RtcB. To capture the step immediately preceding RtcB guanylylation, we solved a structure of RtcB in complex with Mn(II) and the unreactive GTP analogue guanosine 5′-(α-thio)-triphosphate (GTPαS). This structure shows that Mn1 is poised to stabilize the pentavalent transition state of guanylylation while a second manganese ion (Mn2) is coordinated to a nonbridging oxygen of the γ-phosphoryl group. The pyrophosphate leaving group of GTPαS is oriented apically to His404 with the ε nitrogen poised for in-line attack on the α phosphorus atom. The structure of RtcB in complex with GTPαS also reveals the network of hydrogen bonds that recognize GTP and illuminates the significant conformational changes that accompany the binding of this cofactor. Finally, a structure of the enzymic histidine–GMP intermediate depicts the end of the guanylylation pathway. The ensuing molecular description of the RtcB guanylylation pathway shows that RtcB and classical ATP/Mg(II)-dependent nucleic acid ligases have converged upon a similar two-metal mechanism for formation of the nucleotidylated enzyme intermediate.

Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3′-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon.

The identification of novel biomarkers for early prostate cancer diagnosis is highly important because early detection and treatment are critical for the medical management of patients. Disruption in the continuity of both the basal cell layer and basement membrane is essential for the progression of high-grade prostatic intraepithelial neoplasia (HGPIN) to invasive adenocarcinoma in human prostate. The molecules involved in the conversion to an invasive phenotype are the subject of intense scrutiny. We have previously reported that matrix metalloproteinase-26 (MMP-26) promotes the invasion of human prostate cancer cells via the cleavage of basement membrane proteins and by activating the zymogen form of MMP-9. Furthermore, we have found that tissue inhibitor of metalloproteinases-4 (TIMP-4) is the most potent endogenous inhibitor of MMP-26. Here we demonstrate higher (p<0.0001) MMP-26 and TIMP-4 expression in HGPIN and cancer, compared to non-neoplastic acini. Their expression levels are highest in HGPIN, but decline in invasive cancer (p<0.001 for each) in the same tissues. Immunohistochemical staining of serial prostate cancer tissue sections suggests colocalization of MMP-26 and TIMP-4. The present study indicates that MMP-26 and TIMP-4 may play an integral role during the conversion of HGPIN to invasive cancer and may also serve as markers for early prostate cancer diagnosis.

The RNA ligase RtcB is conserved in all domains of life, and is essential for tRNA maturation in archaea and metazoa. Here we show that bacterial and archaeal RtcB catalyze the GTP-dependent ligation of RNA with 3′-phosphate and 5′-hydroxyl termini. Reactions with analogues of RNA and GTP suggest a mechanism in which RtcB heals the 3′-phosphate terminus by forming a 2′,3′-cyclic phosphate before joining it to the 5′-hydroxyl group of a second RNA strand. Thus, RtcB can ligate RNA cleaved by RNA endonucleases, which generate 2′,3′-cyclic phosphate and then 3′-phosphate termini on one strand, and a 5′-hydroxyl terminus on another strand.