Study of the interaction of Escherichia coli methionyl-tRNA synthetase with tRNAfMet using chemical and enzymic probes

Abstract

The accessibility of nucleotides in Escherichia coli tRNAfMet to chemical and enzymatic probes in the presence and absence of methionyl-tRNA synthetase has been investigated. Dimethyl sulfate was used to probe the reactivity of cytosine and guanosine residues. The N-3 position of the wobble anticodon base, C34, was strongly protected from methylation in the tRNA-synthetase complex. A synthetase-induced conformational change in the anticodon loop was suggested by the enhanced reactivity of C32 in the presence of enzyme. Cytosine residues in the dihydrouridine loop and in the 3'-terminal CCA sequence showed little or no change in reactivity. Methylation of the N-7 position of guanosine residues G42, G52, and G70 was partially inhibited by the synthetase. Nuclease digestion of tRNAfMet with alpha-sarcin in the presence of 1-2 mM Mg2+ resulted in cleavage mainly at C71 in the acceptor stem and was strongly inhibited by synthetase. Other nuclease digestion experiments using the single strand specific nucleases RNase A and RNase T1 revealed weak protection of nucleotides in the D loop and strong protection of nucleotides in the anticodon on complex formation. The present data, together with previous structure-function studies on this system, indicate strong binding of methionyl-tRNA synthetase to the anticodon of tRNAfMet, leading to a change in the conformation of the anticodon loop and stem. We propose that this, in turn, produces more distant, and possibly relatively subtle, conformational changes in other parts of the tRNA structure that ultimately lead to proper orientation of the 3' terminus of the tRNA with respect to the active site of the enzyme.

An Operational RNA Code for Faithful Assignment of AUG Triplets to Methionine

“…As expected, the tRNA Met CAU (C34A) wobble base substitution abolishes aminoacylation by both MpMRS (Figure 3B) and EcMRS (data not shown). This confirms the established critical role of this nucleotide in E. coli tRNA Met CAU recognition by EcMRS (Pelka and Schulman, 1986). The tRNA Met CAU (G31A) variant is aminoacylated by MpMRS at a reduced level relative to the wild-type sequence, indicating the importance of the anticodon stem and loop for MpMRS charging efficiency.…”

Molecular mimicry in translational control ofE.colithreonyl-tRNA synthetase gene. Competitive inhibition in tRNA aminoacylation and operator-repressor recognition switch using tRNA identity rules

“…The fact that the anticodon-like domain of BS4-9 is shielded by MetRS but not by ThrRS (5) strongly suggests the existence of specific contacts between the anticodon-like CAU sequence and MetRS. This result is well correlated with the fact that methionine acceptance can be conferred to different tRNA species by inserting a CAU anticodon (15, 24,25), and that the enzyme tightly binds to C34 at the wobble position of the initiator tRNAfMet (24,26,27). Our results show that ThrRS (5) and MetRS both shield the anticodon-like region as well as the acceptor-like domain of the wild type and switched thrS mRNA, respectively.…”

Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNATrp aminoacylation

“…Another possibility is that a conformation change of either macromolecule be limiting. Such a conformational change has been observed during the aminoacylation reaction for several synthetases (Riesner et al, 1978;Kern & Gangloff, 1981;Holler et al, 1981; Baltzinger & Holler, 1982b;Beresten et al, 1983;Ferguson & Yang, 1986a) and for tRNA (Renaud et al, 1981;Lefevre et al, 1981;Yamashiro-Matsumara & Kawata, 1981;Ferguson & Yang, 1986b;Pelka & Schulman, 1986). It may be limiting (Baltzinger & Holler, 1982a).…”