Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editing by Escherichia coli isoleucyl-tRNA synthetase (IleRS). We demonstrate that tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Measurements of the microscopic rate constants for amino acid transfer to tRNA in IleRS and the related valyl-tRNA synthetase (ValRS) further suggest that pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. In this model, the balance between pretransfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate. Rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS, whereas in ValRS transfer to tRNA is 200-fold faster than hydrolysis. In consequence, editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain, whereas in IleRS both pre-and post-transfer editing are important. In both enzymes, the rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates, providing a significant contrast with editing DNA polymerases.
The
accurate expression of genetic information relies on the fidelity
of amino acid–tRNA coupling by aminoacyl-tRNA synthetases (aaRS).
When the specificity against structurally similar noncognate amino
acids in the synthetic reaction does not support a threshold fidelity
level for translation, the aaRS employ intrinsic hydrolytic editing
to correct errors in aminoacylation. Escherichia coli isoleucyl-tRNA synthetase (EcIleRS) is a class I aaRS that is notable
for its use of tRNA-dependent pretransfer editing to hydrolyze noncognate
valyl-adenylate prior to aminoacyl-tRNA formation. On the basis of
the finding that IleRS possessing an inactivated post-transfer editing
domain is still capable of robust tRNA-dependent editing, we have
recently proposed that the pretransfer editing activity resides within
the synthetic site. Here we apply an improved methodology that allows
quantitation of the AMP fraction that arises particularly from tRNA-dependent
aa-AMP hydrolysis. By this approach, we demonstrate that tRNA-dependent
pretransfer editing accounts for nearly one-third of the total proofreading
by EcIleRS and that a highly conserved tyrosine within the synthetic
site modulates both editing and aminoacylation. Therefore, synthesis
of aminoacyl-tRNA and hydrolysis of aminoacyl-adenylates employ overlapping
amino acid determinants. We suggest that this overlap hindered the
evolution of synthetic site-based pretransfer editing as the predominant
proofreading pathway, because that activity is difficult to accommodate
in the context of efficient aminoacyl-tRNA synthesis. Instead, the
acquisition of a spatially separate domain dedicated to post-transfer
editing alone allowed for the development of a powerful deacylation
machinery that effectively competes with dissociation of misacylated
tRNAs.
Isoleucyl-tRNA synthetase (IleRS) is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. Alongside the typical bacterial IleRS (such as Escherichia coli IleRS), some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. Our phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. We present the analysis of evolutionary constraints of the synthetic and editing reactions in eukaryotic/eukaryote-like IleRSs, which share a common origin but diverged through adaptation to different cell environments. The enzyme from the yeast cytosol exhibits tRNA-dependent pre-transfer editing analogous to E. coli IleRS. This argues for the presence of this proofreading in the common ancestor of both IleRS types and an ancient origin of the synthetic site-based quality control step. Yet surprisingly, the eukaryote-like enzyme from Streptomyces griseus IleRS lacks this capacity; at the same time, its synthetic site displays the 103-fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. The discovery that pre-transfer editing is optional in IleRSs lends support to the notion that the conserved post-transfer editing domain is the main checkpoint in these enzymes. We substantiated this by showing that under error-prone conditions S. griseus IleRS is able to rescue the growth of an E. coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability.
Fluorine being not
substantially present in the chemistry of living
beings is an attractive element in tailoring novel chemical, biophysical,
and pharmacokinetic properties of peptides and proteins. The hallmark
of ribosome-mediated artificial amino acid incorporation into peptides
and proteins is a broad substrate tolerance, which is assumed to rely
on the absence of evolutionary pressure for efficient editing of artificial
amino acids. We used the well-characterized editing proficient isoleucyl-tRNA
synthetase (IleRS) from Escherichia coli to investigate
the crosstalk of aminoacylation and editing activities against fluorinated
amino acids. We show that translation of trifluoroethylglycine (TfeGly)
into proteins is prevented by hydrolysis of TfeGly-tRNAIle in the IleRS post-transfer editing domain. The remarkable observation
is that dissociation of TfeGly-tRNAIle from IleRS is significantly
slowed down. This finding is in sharp contrast to natural editing
reactions by tRNA synthetases wherein fast editing rates for the noncognate
substrates are essential to outcompete fast aa-tRNA dissociation rates.
Using a post-transfer editing deficient mutant of IleRS (IleRSAla10),
we were able to achieve ribosomal incorporation of TfeGly in vivo.
Our work expands the knowledge of ribosome-mediated artificial amino
acid translation with detailed analysis of natural editing function
against an artificial amino acid providing an impulse for further
systematic investigations and engineering of the translation and editing
of unusual amino acids.
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