Protein Synthesis in Escherichia coli with Mischarged tRNA

Abstract

Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA Asp , while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA Asn , a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA Asn and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA Asn are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.Aspartyl-tRNA synthetase (AspRS) exists in two different forms with respect to tRNA recognition (7). The discriminating enzyme (D-AspRS) recognizes only tRNA Asp , while the nondiscriminating one (ND-AspRS) also recognizes tRNA Asn and therefore forms both Asp-tRNA Asn and Asp-tRNA Asp . Most bacteria and archaea lack asparaginyl-tRNA synthetase and are unable to synthesize Asn-tRNA Asn by direct acylation of tRNA. These organisms rely on the ND-AspRS to produce the misacylated Asp-tRNA Asn , which is then converted by a tRNA-dependent amidotransferase to the correctly acylated Asn-tRNA Asn (1,4,5,19). Thus, the ND-AspRS is essential in organisms that form Asn-tRNA by transamidation.The primary sequence distinguishes two general groups of AspRS. There is a predominantly bacterial type of AspRS that is about 580 amino acids, in addition to a shorter archaealeukaryotic type of about 430 amino acids. In vitro data have made clear that discriminating and nondiscriminating enzymes exist in both groups (16,20). The determinants in the protein sequence responsible for tRNA discrimination are not known.The two AspRS types are usually separated in nature. Genome analyses of bacteria and archaea have revealed that the presence of the ND-AspRS is always accompanied by the occurrence of the heterotrimeric GatCAB amidotransferase, an enzyme capable of converting the misacylated Asp-tRNA Asn to Asn-tRNA Asn (2,5,19). Presumably, this is to avoid introducing the misacylated Asp-tRNA Asn into an organism's translational apparatus and potentially endangering protein synthesis. This reasoning is supported by the fact that the heterologous expression of ND-AspRS or ND-GluRS in Escherichia coli, which lacks GatCAB, is highly toxic to the cell, especially when the synthetase genes are overexpressed (15). However, some organisms (e.g., Deinococcus radiodurans and Thermus thermophilus) contain a D-AspRS in addition to an ND-AspRS and a GatCAB amidotransferase (1,3,5,9).We wanted to observe how E. coli copes with in vivo mischarging effected by the ND-AspRS, as this organism is unable to eliminate the toxic Asp-tRNA Asn . Therefore, we developed an approach that would, in fact, require E. coli to be dependent on the presence of mischarged Asp-tRNA Asn for growth. To this aim, we used missense suppression of a specific mutation in the trp...

Quality control despite mistranslation caused by an ambiguous genetic code

“…17) is also more widespread than previously appreciated. The in vivo presence of misacylated aa-tRNA introduces ambiguity into the genetic code and leads to the generation of ''statistical'' proteins (18), whereby the use of misacylated in addition to correctly charged aa-tRNA by the ribosome during translation results in a group of related proteins for any given messenger RNA.…”

“…This system was tested in the E. coli trpA34 background (17,38). After transformation with pCBSaspS2, single colonies, streaked on medium B agar plates in the presence or absence of Trp, were incubated at 37°C.…”

The Nondiscriminating Aspartyl-tRNA Synthetase from Helicobacter pylori: Anticodon-Binding Domain Mutations That Impact tRNA Specificity and Heterologous Toxicity

“…At least in some organisms, EF-Tu plays a direct role in preventing Asp-tRNA Asn and Glu-tRNA Gln from entering the ribosome and causing errors in protein biosynthesis (52,29,3), arguing that this toxicity arises from sequestration of tRNA Asn as the abortive Asp-tRNA Asn . However, successful selection experiments in E. coli have relied on Asp-tRNA Asn -mediated incorporation of Asp into proteins at Asn codons, arguing that E. coli EF-Tu binds this misacylated tRNA at least to an extent to be functionally significant (11,33). Thus, it remains to be seen whether the toxicity of Hp ND-AspRS is due to a dearth of Asn-tRNA Asn or to the induction of catastrophic errors arising from low levels of Asp for Asn mutations at a proteome-wide level.…”

Aminoacyl-tRNAs: setting the limits of the genetic code

“…The next step in protein synthesis requires the association of aa-tRNAs with translation factors, and this is used to provide an additional checkpoint. Although EF-Tu discrimination is not absolute (Min et al 2003;Nú ñ ez et al 2004), it can distinguish both precursor noncognate tRNAs (see above) and other incorrectly charged tRNA species ; Asahara and Uhlenbeck 2002), whereas initiation factor 2 specifically binds initiator aa-tRNAs (Mayer et al 2003). Finally, it has been suggested that the ribosome itself might also provide some degree of selectivity towards cognate aa-tRNAs .…”

“…More often, however, the unintended synthesis of noncognate aa-tRNAs presents a threat to the accuracy of translation. Consequently a variety of quality control strategies are employed by the cell to ensure that typically only about one in every 10 4 codons is mistranslated, even though significantly higher rates at particular codons can be tolerated (Ruusala et al 1982;Kurland 1992;Min et al 2003;Nú ñ ez et al 2004). Aside from codon-anticodon pairing (Ogle et al 2003), the mechanisms of translational quality control are broadly of three types; specificity of substrate selection by aa-tRNA synthetases, proofreading, and exclusion from the ribosome.…”