Differential Modes of Transfer RNASer Recognition in Methanosarcina barkeri

Abstract

Two dissimilar seryl-transfer RNA (tRNA) synthetases (SerRSs) exist in Methanosarcina barkeri, one of bacterial type and the other resembling SerRSs present only in some methanogenic archaea. To investigate the requirements of these enzymes for tRNA Ser recognition, serylation of variant transcripts of M. barkeri tRNA Ser was kinetically analyzed in vitro with pure enzyme preparations. Characteristically for the serine system, the length of the variable arm was shown to be crucial for both enzymes, as was the identity of the discriminator base (G73). Moreover, a novel determinant for the specific tRNA Ser recognition was identified as the anticodon stem base pair G30:C40; its contribution to the efficiency of serylation was remarkable for both SerRSs. However, despite these similarities, the two SerRSs do not possess a uniform mode of tRNA Ser recognition, and additional determinants are necessary for serylation specificity by the methanogenic enzyme. In particular, the methanogenic SerRS relies on G1:C72 identity and on the number of unpaired nucleotides at the base of the variable stem for tRNA Ser recognition, unlike its bacterial type counterpart. We propose that such a distinction between the two enzymes in tRNA Ser identity determinants reflects their evolutionary pathways, hence attesting to their diversity.To maintain translational accuracy, aminoacyl-transfer RNA (tRNA) 1 synthetases are highly selective toward their amino acid and tRNA substrates. In the process of tRNA recognition, the cognate and non-cognate substrates are discriminated according to characteristic nucleotides in certain positions of the tRNA, specific for the tRNA/synthetase system.

Molecular Evolution of Aminoacyl tRNA Synthetase Proteins in the Early History of Life

“…Despite their differences, the rare and common forms perform identical functions, aminoacylating serine and ligating it to its cognate tRNA molecule. In rare instances, both forms are found in the same genome, as in the case of Methanosarcina barkeri (Korencic et al 2004;Andam and Gogarten 2011a), which would suggest the acquisition of a second but divergent form of the enzyme by a genome already possessing an initial copy.…”

“…The rare form does not exhibit high sequence or structural similarity to other SerRS found in the majority of organisms within the three domains of life (Kim et al 1998;Andam and Gogarten 2011a). Another significant difference between the two forms is their mechanism of substrate recognition (Korencic et al 2004). Although both reveal some similarities in their mode of tRNA Ser recognition, there are remarkable differences in their identity requirements, such as the G1:C72 base pair and the number of unpaired nucleotides at the base of the variable stem that are both required by the rare SerRS (Korencic et al 2004).…”

“…Another significant difference between the two forms is their mechanism of substrate recognition (Korencic et al 2004). Although both reveal some similarities in their mode of tRNA Ser recognition, there are remarkable differences in their identity requirements, such as the G1:C72 base pair and the number of unpaired nucleotides at the base of the variable stem that are both required by the rare SerRS (Korencic et al 2004). Serine recognition of the rare form is also dependent on a zinc ion present in the active site, which is not found in the common SerRS (Bilokapic et al 2006).…”

Emergence of the universal genetic code imprinted in an RNA record

“…The discriminator base is not an identity element in this case (51). Both SerRSs share major identity elements in the extra arm, discriminator G73 and the G30:C40 base pair, whereas the rare archaeal form has G1:C72 and additional unpaired bases in the extra arm as additional identity elements (52). Although tRNA Ser from Me.…”

Substrate Recognition by Novel Family of Amino Acid:[Carrier Protein] Ligases

“…21 PCR fragments coding for fusion proteins were cloned to pET28b, and N-terminal His-tagged fusion proteins were overexpressed and purified as described. Preparation of SerRS from E. coli 22 and M. barkeri 23 was described previously. Isolation of unfractioned tRNA from B. japonicum and A. tumefaciens was performed by phenol extraction and polyetilene-glycol precipitation.…”