Effects of Mutagenesis in the Switch I Region and Conserved Arginines of Escherichia coli MnmE Protein, A GTPase Involved in tRNA Modification

Abstract

MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif ( 249 GTTRD 253 ), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly 249 and Thr 251 . On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr 251 ) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.

Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification

“…Capability of proteins to bind guanine nucleotides was analyzed by using fluorescent nucleotides as described previously (27). The K d values of GST-MnmE for mant GPT␥S (mGTP␥S) and mant GDP (mGDP) were similar to those of MnmE and remained in the low micromolar range, which indicates that the GST moiety does not significantly alter the nucleotide binding properties of MnmE (Table 3).…”

“…Protein techniques and production of antisera. Expression and purification of the GST-MnmE protein was performed as previously described (27). Overproduction of GST-fused GTPBP3 was done in DH5␣ cells transformed with pIC1298 or pIC1299.…”

“…This is particularly interesting because exon 8A encodes 21 amino acids located between the G3 and G4 motifs of the GTPBP3 G domain. Since the GTPase activity of MnmE is essential for its tRNA modification function (27,57), the lack of exon 8A could have significant consequences for the functionality of the pro- tein encoded by variant III. Variant IV carries an additional exon 7B at the beginning of cDNA and lacks exons 1A and 8A.…”

“…These properties determine that, in vitro, the GTPase cycle of MnmE proceeds efficiently without auxiliary factors (i.e., GTPase-activating proteins and guanine nucleotide exchange factors). Interestingly, the GTPase activity is essential for the tRNA modifying function of MnmE (27,57).…”

Interactome analysis of longitudinal pharyngeal infection of cynomolgus macaques by group A Streptococcus

“…For example, the GAS thdF gene had an inverse correlation with PLG expression. Although little is known about the function of many of these GTPases, thdF catalyzes the modification of certain tRNA species and is required for proper base-pairing during codon-anticodon recognition (21,22). Inactivation of these genes induces frame-shifting and premature termination during translation (23), particularly affecting proteins with extensive regions of positive charge, such as DNA-binding transcription factors.…”