Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Tamara L. Hendrickson

Abstract

H. pylori is missing the glutaminyl- and asparaginyl-tRNA synthetases (GlnRS and AsnRS, respectively). Consequently, H. pylori uses an indirect aminoacylation pathway to generate Gln-tRNAGln and Asn-tRNAAsn. Within this process, Asn-tRNAAsn is produced by misacylation of tRNAAsn with aspartate by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS). Next, the heterotrimeric, glutamine-dependent amidotransferase (called AdT or GatCAB) converts the misacylated Asp-tRNAAsn into Asn-tRNAAsn. A parallel pathway exists for the synthesis of Gln-tRNAGln, wherein misacylation of tRNAGln with glutamate is catalyzed by a tRNAGln-specific glutamyl-tRNA synthetase (GluRS2) to generate Glu-tRNAGln; this misacylated intermediate is converted to Gln-tRNAGln by the same AdT. This dependence on misacylated intermediates for protein synthesis suggests a requirement for additional mechanisms to prevent the misincorporation of Glu and Asp into proteins in place of Gln and Asn. H. pylori elongation factor (EF-Tu) provides one such machinery, but it is not sufficient to maintain translational accuracy, suggesting the need for additional mechanisms.

Hp0495 and Hp0100 were identified by yeast two-hybrid (Y2H) as potential new players in tRNA aminoacylation and fidelity. By Y2H, Hp0495 showed interactions with EF-Tu and Hp0100 was connected to both ND-AspRS and AdT. The work presented in this dissertation examined the possible roles of both of these proteins in indirect aminoacylation and in promoting tRNA accuracy.

Using SPR, native gels, and size exclusion chromatography, we have shown that Hp0495 forms complexes with EF-Tu in both its GDP and GTP forms, but preferentially binds EF-Tu*GTP. In collaboration with a colleague, Dr. Keng-Ming Chang, we have discovered that Hp0495 binds ATP, glutamate, and either tRNAGlu1 or tRNAGln and forms tRNAGln-dependent complex with GluRS2. Also, Hp0495 binds deacylated tRNAs more tightly than aminoacylated tRNAs for the five cases tested in this work. Hp0495 has similarities to ACT/RAM domains; these domains typically regulate different aspects of amino acid and nucleotide metabolism. Our hypothesis is that Hp0495 might be a regulatory protein, sensing tRNAGlu1/tRNAGln and directing one or both it to either GluRS1/GluRS2 for aminoacylation or for functions outside protein translation. The hp0495 gene is in an operon with mraY and murD, these two enzymes are involved in cell wall biosynthesis, suggesting that Hp0495 might be involved in regulating this process. There is also precedence for the involvement of EF-Tu in cell wall biosynthesis, supporting this hypothesis.

In collaboration with a colleague, Dr. Gayathri Silva, we have demonstrated that Hp0100 is, in fact, an essential component of a tRNA-independent Asn-transamidosome complex. This complex contains ND-AspRS, AdT, and Hp0100 and facilitates AdT's transamidation process of Asp-tRNAAsn (~ 35-fold) and Glu-tRNAGln (~ 3-fold). Our work characterizing the Asn-transamidosome and transamidation of Asp-tRNAAsn was published in 2013 (Silva, G. N., Fatma, S., et al. J. Biol. Chem. 288, 3816). An hp0100 knockout suggests that Hp0100 is not essential under robust growth conditions. Our revised hypothesis is that the growth conditions used in these experiments were too permissive and did not adequately reflect the stress conditions faced by H. pylori in its human niche. Viability of our hp0100 strain will be tested under different stress conditions with the goal of identifying conditions where Hp0100 is conditionally essential.

Finally, we have initiated the characterization of third protein of unknown function, Hp1259. Preliminary data suggest that it might be involved in the indirect aminoacylation of tRNAGln.

In the H. pylori genome, 499 open reading frames (ORFs) are annotated as hypothetical proteins whose functions are not known, including Hp0100 and Hp0495. We have assigned an important function to Hp0100, namely promoting tRNA aminoacylation, and have identified a new functional characteristics for Hp0495 that clearly demonstrate its role in tRNA aminoacylation and/or function. The existence of these proteins of unknown function and their interactions with components of the translation machinery clearly demonstrate that the classical boundaries of the field of tRNA aminoacylation need to be expanded.

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