Editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for accurate translation of the genetic code. To date, this activity, whereby misactivated amino acids are hydrolyzed either before or after transfer to noncognate tRNAs, has been characterized extensively only in the case of class I synthetases. Class II synthetases have an active-site architecture that is completely distinct from that of class I. Thus, findings on editing by class I synthetases may not be applicable generally to class II enzymes. Class II Escherichia coli proline-tRNA synthetase is shown here to misactivate alanine and to hydrolyze the noncognate amino acid before transfer to tRNA Pro . This enzyme also is capable of rapidly deacylating a mischarged Ala-tRNA Pro variant. A single cysteine residue (C443) that is located within the class II-specific motif 3 consensus sequence was shown previously to be dispensable for proline-tRNA synthetase aminoacylation activity. We show here that C443 is critical for the hydrolytic editing of Ala-tRNA Pro by this class II synthetase.

The observation that mutations in the Escherichia coli genes umuC+ and umuD+ abolish mutagenesis induced by UV light strongly supported the counterintuitive notion that such mutagenesis is an active rather than passive process. Genetic and biochemical studies have revealed that umuC+ and its homolog dinB+ encode novel DNA polymerases with the ability to catalyze synthesis past DNA lesions that otherwise stall replication--a process termed translesion synthesis (TLS). Similar polymerases have been identified in nearly all organisms, constituting a new enzyme superfamily. Although typically viewed as unfaithful copiers of DNA, recent studies suggest that certain TLS polymerases can perform proficient and moderately accurate bypass of particular types of DNA damage. Moreover, various cellular factors can modulate their activity and mutagenic potential.

Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to cognate tRNAs in a two-step process that is critical for the faithful translation of genetic information. During the first chemical step of tRNA aminoacylation, noncognate amino acids that are smaller than or isosteric with the cognate substrate can be misactivated. Thus, to maintain high accuracy during protein translation, some synthetases have evolved an editing mechanism. Previously, we showed that class II Escherichia coli proline-tRNA synthetase (ProRS) is capable of (1) weakly misactivating Ala, (2) hydrolyzing the misactivated Ala-AMP in a reaction known as pretransfer editing, and (3) deacylating a mischarged Ala-tRNA(Pro) variant via a post-transfer editing pathway. In contrast to most systems where an editing function has been established, pretransfer editing by E. coli ProRS occurs in a tRNA-independent fashion. However, neither the pre- nor the post-transfer editing active site(s) has been identified. Sequence analyses revealed that most prokaryotic ProRSs possess a large insertion domain (INS) between class II conserved motifs 2 and 3. The function of the approximately 180-amino acid INS in E. coli ProRS is the subject of this investigation. Alignment-guided Ala scanning mutagenesis was carried out to test conserved amino acid residues present in the INS for their role in pre- and post-transfer editing. Our biochemical data and modeling studies suggest that the prokaryotic INS plays a critical role in editing and that this activity resides in a domain that is functionally and structurally distinct from the aminoacylation active site.

Discovery of EX1 kinetics in hydrogen exchange (HX) mass spectrometry (MS) experiments is rare. Proteins follow the EX1 kinetic regime when cooperative unfolding events simultaneously expose multiple residues to solvent such that they all become deuterated together before the region is able to refold. A number of factors can contribute to what we call "false EX1" in which it appears as though EX1 occurs in a protein when it probably does not. One of the contributors to false EX1 is peptide carryover between chromatographic runs. In this work, we explore the origins of peptide carryover in HX MS, describe how carryover causes mass spectra to indicate false EX1 kinetics and then describe an optimized washing protocol that can be used to eliminate peptide carryover. A series of solvent injections was developed and found to efficiently eliminate carryover signatures such that analysis of deuterium incorporation could be reliably followed for two proteins prone to high carryover.

UmuD 2 cleaves and removes its N-terminal 24 amino acids to form UmuD 2 , which activates UmuC for its role in UV-induced mutagenesis in Escherichia coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD binds to the ␤ processivity clamp ("␤") of the replicative DNA polymerase, pol III. A possible ␤-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with ␤. We performed alanine-scanning mutagenesis of this motif ( 14 TFPLF 18 ) in UmuD and found that it has a moderate influence on UV-induced mutagenesis but is required for the cold-sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the ␤-binding motif variant bind to ␤ with similar K d values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in ␤ is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is non-cleavable, we find that cells harboring it display phenotypes more consistent with the cleaved form UmuD, such as resistance to killing by UV light and failure to exhibit the cold-sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD mimic.The umuDC gene products are induced as part of the SOS response and are responsible for much of the UV-induced mutagenesis in Escherichia coli (1). These gene products are subject to an elaborate set of controls that regulate their activity (1). The LexA repressor provides transcriptional control, and there are several proteolytic controls on both the umuD and umuC gene products (1). The homodimeric protein UmuD 2 is the predominant species during the first about 20 -30 min after SOS induction (2). UmuD 2 , together with UmuC, plays a role in a DNA damage checkpoint, decreasing the rate of DNA synthesis and allowing time for accurate repair processes to act (2). This correlates with the cold-sensitive phenotype observed under conditions of overexpression of the umuDC gene products (2, The wealth of structural data and models available for UmuD 2 and UmuDЈ 2 provide insight into how the two forms of the umuD gene products engage in multiple highly specific interactions ( Fig. 1) (4 -8), including with the ␣, ␤, and ⑀ subunits of the replicative polymerase, pol III (9). Of the two forms, UmuD 2 interacts more strongly with the ␤ processivity clamp (also referred to as ␤ or the ␤ clamp) than does UmuDЈ 2 (9, 10). In full-length UmuD 2 , the 39-amino acid N-terminal arms are stably bound to the globular C-terminal domain (4, 7) and form a distinct interaction surface. In t...

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Aminoacyl-tRNA synthetases are responsible for activating specific amino acids and transferring them onto cognate tRNA molecules. Due to the similarity in many amino acid side chains, certain synthetases misactivate non-cognate amino acids to an extent that would be detrimental to protein synthesis if left uncorrected. To ensure accurate translation of the genetic code, some synthetases therefore utilize editing mechanisms to hydrolyze non-cognate products. Previously class II Escherichia coli proline-tRNA synthetase (ProRS) was shown to exhibit pre-and post-transfer editing activity, hydrolyzing a misactivated alanine-adenylate (Ala-AMP) and a mischarged Ala-

Aminoacyl-tRNA synthetases are a family of enzymes responsible for ensuring the accuracy of the genetic code by specifically attaching a particular amino acid to their cognate tRNA substrates. Through primary sequence alignments, prolyl-tRNA synthetases (ProRSs) have been divided into two phylogenetically divergent groups. We have been interested in understanding whether the unusual evolutionary pattern of ProRSs corresponds to functional differences as well. Previously, we showed that some features of tRNA recognition and aminoacylation are indeed groupspecific. Here, we examine the species-specific differences in another enzymatic activity, namely amino acid editing. Proofreading or editing provides a mechanism by which incorrectly activated amino acids are hydrolyzed and thus prevented from misincorporation into proteins. "Prokaryotic-like" Escherichia coli ProRS has recently been shown to be capable of misactivating alanine and possesses both pretransfer and post-transfer hydrolytic editing activity against this noncognate amino acid. We now find that two ProRSs belonging to the "eukaryotic-like" group exhibit differences in their hydrolytic editing activity. Whereas ProRS from Methanococcus jannaschii is similar to E. coli in its ability to hydrolyze misactivated alanine via both pretransfer and post-transfer editing pathways, human ProRS lacks these activities. These results have implications for the selection or design of antibiotics that specifically target the editing active site of the prokaryotic-like group of ProRSs.