Tamara L. Hendrickson

Transfer RNA–Dependent Translocation of Misactivated Amino Acids to Prevent Errors in Protein Synthesis

Misactivation of amino acids by aminoacyl-tRNA synthetases can lead to significant errors in protein synthesis that are prevented by editing reactions. As an example, discrete sites in isoleucyl-tRNA synthetase for amino acid activation and editing are about 25 A apart. The details of how misactivated valine is translocated from one site to the other are unknown. Here, we present a kinetic study in which a fluorescent probe is used to monitor translocation of misactivated valine from the active site to the editing site. Isoleucine-specific tRNA, and not other tRNAs, is essential for translocation of misactivated valine. Misactivation and translocation occur on the same enzyme molecule, with translocation being rate limiting for editing. These results illustrate a remarkable capacity for a specific tRNA to enhance amino acid fine structure recognition by triggering a unimolecular translocation event.

A noncognate aminoacyl-tRNA synthetase that may resolve a missing link in protein evolution

Efforts to delineate the advent of many enzymes essential to protein translation are often limited by the fact that the modern genetic code evolved before divergence of the tree of life. Glutaminyl-tRNA synthetase (GlnRS) is one noteworthy exception to the universality of the translation apparatus. In eukaryotes and some bacteria, this enzyme is essential for the biosynthesis of Gln-tRNAGln, an obligate intermediate in translation. GlnRS is absent, however, in archaea, and most bacteria, organelles, and chloroplasts. Phylogenetic analyses predict that GlnRS arose from glutamyl-tRNA synthetase (GluRS), via gene duplication with subsequent evolution of specificity. A pertinent question to ask is whether, in the advent of GlnRS, a transient GluRS-like intermediate could have been retained in an extant organism. Here, we report the discovery of an essential GluRS-like enzyme (GluRS2), which coexists with another GluRS (GluRS1) in Helicobacter pylori. We show that GluRS2s primary role is to generate Glu-tRNAGln, not Glu-tRNAGlu. Thus, GluRS2 appears to be a transient GluRS-like ancestor of GlnRS and can be defined as a GluGlnRS.

Mutational Separation of Two Pathways for Editing by a Class I tRNA Synthetase

Aminoacyl tRNA synthetases (aaRSs) catalyze the first step in protein biosynthesis, establishing a connection between codons and amino acids. To maintain accuracy, aaRSs have evolved a second active site that eliminates noncognate amino acids. Isoleucyl tRNA synthetase edits valine by two tRNA(Ile)-dependent pathways: hydrolysis of valyl adenylate (Val-AMP, pretransfer editing) and hydrolysis of mischarged Val-tRNA(Ile) (posttransfer editing). Not understood is how a single editing site processes two distinct substrates--an adenylate and an aminoacyl tRNA ester. We report here distinct mutations within the center for editing that alter adenylate but not aminoacyl ester hydrolysis, and vice versa. These results are consistent with a molecular model that shows that the single editing active site contains two valyl binding pockets, one specific for each substrate.

Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where GluRS2 hydrolyzes excess Glu-tRNAGln

In many bacteria and archaea, an ancestral pathway is used where asparagine and glutamine are formed from their acidic precursors while covalently linked to tRNAAsn and tRNAGln, respectively. Stable complexes formed by the enzymes of these indirect tRNA aminoacylation pathways are found in several thermophilic organisms, and are called transamidosomes. We describe here a transamidosome forming Gln-tRNAGln in Helicobacter pylori, an ε-proteobacterium pathogenic for humans; this transamidosome displays novel properties that may be characteristic of mesophilic organisms. This ternary complex containing the non-canonical GluRS2 specific for Glu-tRNAGln formation, the tRNA-dependent amidotransferase GatCAB and tRNAGln was characterized by dynamic light scattering. Moreover, we observed by interferometry a weak interaction between GluRS2 and GatCAB (KD = 40 ± 5 µM). The kinetics of Glu-tRNAGln and Gln-tRNAGln formation indicate that conformational shifts inside the transamidosome allow the tRNAGln acceptor stem to interact alternately with GluRS2 and GatCAB despite their common identity elements. The integrity of this dynamic transamidosome depends on a critical concentration of tRNAGln, above which it dissociates into separate GatCAB/tRNAGln and GluRS2/tRNAGln complexes. Ester bond protection assays show that both enzymes display a good affinity for tRNAGln regardless of its aminoacylation state, and support a mechanism where GluRS2 can hydrolyze excess Glu-tRNAGln, ensuring faithful decoding of Gln codons.

Chemistry and biology of asparagine-linked glycosylation

The biosynthesis of glycoprotein conjugates is a complex process that involves the collective action of numerous enzymes. Recent research on the chemistry and biology of asparagine-linked glycosylation in our group has been focused on two specific areas. These are the development of potent inhibitors of oligosaccharyl transferase and the investigation of the conformational consequences of the glycosylation process. Since asparagine-linked glycosylation is an essential eukaryotic process, an understanding of the details of this complex transformation is of utmost importance both to fundamental biochemistry and to a consideration of the mechanisms of homeostatic control.

GPI Transamidase and GPI anchored proteins: Oncogenes and biomarkers for cancer

Abstract Cancer is second only to heart disease as a cause of death in the US, with a further negative economic impact on society. Over the past decade, details have emerged which suggest that different glycosylphosphatidylinositol (GPI)-anchored proteins are fundamentally involved in a range of cancers. This post-translational glycolipid modification is introduced into proteins via the action of the enzyme GPI transamidase (GPI-T). In 2004, PIG-U, one of the subunits of GPI-T, was identified as an oncogene in bladder cancer, offering a direct connection between GPI-T and cancer. GPI-T is a membrane-bound, multi-subunit enzyme that is poorly understood, due to its structural complexity and membrane solubility. This review is divided into three sections. First, we describe our current understanding of GPI-T, including what is known about each subunit and their roles in the GPI-T reaction. Next, we review the literature connecting GPI-T to different cancers with an emphasis on the variations in GPI-T subunit over-expression. Finally, we discuss some of the GPI-anchored proteins known to be involved in cancer onset and progression and that serve as potential biomarkers for disease-selective therapies. Given that functions for only one of GPI-T’s subunits have been robustly assigned, the separation between healthy and malignant GPI-T activity is poorly defined.

Methods to study GPI anchoring of proteins.

Glycosylphosphatidylinositol (GPI)-anchored proteins are localized on the surface of eukaryotic cells where they carry out many important functions. Thus GPI transamidase (GPI-T), the enzyme that attaches the GPI anchor to these proteins, is essential for normal cell function. GPI-T is a multisubunit, membrane-bound enzyme; studies of this complicated enzyme and the GPI anchor have relied on the interplay of multiple disciplines, ranging from cell biology to synthetic chemistry, and the adaptation of a wide variety of experimental techniques. Here, we review the methods that have been used to study GPI-T to date and draw attention to the major technical hurdles that remain if we are to understand the complexity of this enzyme. The continued study of GPI anchoring is essential to understand the role(s) of this modification in oncogenesis and as a potential therapeutic target for diseases like malaria and leishmaniasis. 3] Continued studies are also likely to expand the toolbox of techniques available for studying other membrane-bound protein complexes.

Conserved discrimination against misacylated tRNAs by two mesophilic elongation factor Tu orthologs.

Elongation factor Tu (EF-Tu) binds and loads elongating aminoacyl-tRNAs (aa-tRNAs) onto the ribosome for protein biosynthesis. Many bacteria biosynthesize Gln-tRNA (Gln) and Asn-tRNA (Asn) by an indirect, two-step pathway that relies on the misacylated tRNAs Glu-tRNA (Gln) and Asp-tRNA (Asn) as intermediates. Previous thermodynamic and experimental analyses have demonstrated that Thermus thermophilus EF-Tu does not bind Asp-tRNA (Asn) and predicted a similar discriminatory response against Glu-tRNA (Gln) [Asahara, H., and Uhlenbeck, O. (2005) Biochemistry 46, 6194-6200; Roy, H., et al. (2007) Nucleic Acids Res. 35, 3420-3430]. By discriminating against these misacylated tRNAS, EF-Tu plays a direct role in preventing misincorporation of aspartate and glutamate into proteins at asparagine and glutamine codons. Here we report the characterization of two different mesophilic EF-Tu orthologs, one from Escherichia coli, a bacterium that does not utilize either Glu-tRNA (Gln) or Asp-tRNA (Asn), and the second from Helicobacter pylori, an organism in which both misacylated tRNAs are essential. Both EF-Tu orthologs discriminate against these misacylated tRNAs, confirming the prediction that Glu-tRNA (Gln), like Asp-tRNA (Asn), will not form a complex with EF-Tu. These results also demonstrate that the capacity of EF-Tu to discriminate against both of these aminoacyl-tRNAs is conserved even in bacteria like E. coli that do not generate either misacylated tRNA.

Recognizing the D-loop of transfer RNAs

Mechanisms that maintain fidelity and repair mistakes are ubiquitous throughout the protein biosynthesis pathway (1). The aminoacyl tRNAs serve as critical turning points in translation, because they link the nucleic acid genetic code with the amino acid building blocks of proteins. Misacylation of tRNAs can have devastating results, affecting the very survivability of an organism. Accuracy in tRNA aminoacylation therefore is paramount to the fidelity of the genetic code. Aminoacyl tRNAs are generated typically through action of the diverse family of aminoacyl-tRNA synthetases (AARSs). One of the hallmarks of these enzymes is the exquisite specificity with which each selects and aminoacylates only its cognate tRNA(s) with only its cognate amino acid (2–5). Herculean efforts spanning nearly 25 years have produced crystal structures for 19 of the 20 AARSs (alanyl-tRNA synthetase is the last holdout). Many of these structures provide detailed molecular insight into the nature of tRNA recognition and discrimination by either providing a structure of the enzyme in complex with its cognate tRNA or suggesting a model that can be tested biochemically. Thus, a clear picture is beginning to emerge, delineating some of the common and not-so-common themes used by the AARSs to discriminate cognate from noncognate tRNAs. [There are several reviews available that describe tRNA recognition by AARSs in detail (4–7).]

A tRNA-Independent Mechanism for Transamidosome Assembly Promotes Aminoacyl-tRNA Transamidation

Background: Some microorganisms use indirect tRNA aminoacylation to produce Asn-tRNAAsn; the necessary components are assembled into a tRNAAsn-dependent transamidosome complex. Results: A new protein, Hp0100, facilitates formation of an alternative, tRNA-independent transamidosome and increases the efficiency of Asp-tRNAAsn transamidation. Conclusion: Hp0100 is a component of a stable efficient Helicobacter pylori transamidosome. Significance: The Hp0100-containing transamidosome allows for optimal indirect biosynthesis of Asn-tRNAAsn. Many bacteria lack genes encoding asparaginyl- and/or glutaminyl-tRNA synthetase and consequently rely on an indirect path for the synthesis of both Asn-tRNAAsn and Gln-tRNAGln. In some bacteria such as Thermus thermophilus, efficient delivery of misacylated tRNA to the downstream amidotransferase (AdT) is ensured by formation of a stable, tRNA-dependent macromolecular complex called the Asn-transamidosome. This complex enables direct delivery of Asp-tRNAAsn from the non-discriminating aspartyl-tRNA synthetase to AdT, where it is converted into Asn-tRNAAsn. Previous characterization of the analogous Helicobacter pylori Asn-transamidosome revealed that it is dynamic and cannot be stably isolated, suggesting the possibility of an alternative mechanism to facilitate assembly of a stable complex. We have identified a novel protein partner called Hp0100 as a component of a stable, tRNA-independent H. pylori Asn-transamidosome; this complex contains a non-discriminating aspartyl-tRNA synthetase, AdT, and Hp0100 but does not require tRNAAsn for assembly. Hp0100 also enhances the capacity of AdT to convert Asp-tRNAAsn into Asn-tRNAAsn by ∼35-fold. Our results demonstrate that bacteria have adopted multiple divergent methods for transamidosome assembly and function.