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

ABSTRACT

ASSEMBLY AND FUNCTION OF MACROMOLECULAR COMPLEXES FOR ACCURATE TRNA AMINOACYLATION IN HELICOBACTER PYLORI

by

GAYATHRI SILVA

January 2014

Advisor: Dr. Tamara L. Hendrickson

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Abstract

The aminoacylation of tRNA is a critical step in maintaining the accuracy of the genetic code. Many microorganisms are missing one or more aminoacyl tRNA synthetases (aaRSs) and rely on indirect pathways to produce certain aa–tRNAs. In Helicobacter pylori (H. pylori), the genes encoding both asparaginyl tRNA synthetase (AsnRS) and glutaminyl tRNA synthetase (GlnRS) are missing and the organism consequently relies on the indirect pathway for the synthesis of Asn–tRNAAsn and Gln–tRNAGln. The first step of indirect synthesis of Asn–tRNAAsn involves misacylation of tRNAAsn by non–discriminating aspartyl tRNA synthetase (ND–AspRS) to produce Asp–tRNAAsn. Next, the misacylated tRNA is converted to Asn–tRNAAsn by Asp–tRNAAsn/Glu–tRNAGln amidotransferase (Asp/Glu–AdT) to produce Asn–tRNAAsn. Gln–tRNAGln is produced vial an analogous process, relying on a misacylating glutamyl tRNA synthetase, GluRS2 and AdT.

Including H. pylori, organisms that indirectly synthesize Asn–tRNAAsn and Gln–tRNAGln require a mechanism for the efficient delivery of both misacylated tRNAs from the two misacylating enzymes to the amidotransferase enzyme. This delivery mechanism should ensure the stability of the aminoacyl ester bond and prevent translational errors. Some bacteria, like Thermus thermophilus (T. thermophilus), utilize a tRNA–dependent ribonucleoprotein complex (RNC) called the transamidosome. The T. thermophilus transamidosome contains AdT, tRNAAsn, and an archaeal ND–AspRS. This Asn–transamidosome traps misacylated Asp–tRNAAsn until it has been converted to Asn–tRNAAsn by AdT. Similarly, the thermophilic archaeon Methanothermobacter thermoautotrophicus utilizes a Gln–transamidosome for the synthesis of Gln–tRNAGln. This complex consists of ND–GluRS, tRNAGln and GatDE. (GatDE is a heterodimeric homolog of AdT.)

In contrast to T. thermophilus, H. pylori utilizes a bacterial ND–AspRS that has an extra domain that could sterically prevent transamidosome assembly. H. pylori also requires AdT for conversion of both Asp–tRNAAsn and Glu–tRNAGln into their cognate aa–tRNAs. In fact the H. pylori Asn– and Gln–transamidosomes were not stably isolated in vitro, suggesting a requirement for an alternative mechanism.

We describe the first characterization of a novel protein called Hp0100 in H. pylori, Hp0100 is required for the assembly of a stable, tRNA–independent Asn–transamidosome, consisting of ND–AspRS, AdT and Hp0100. Hp0100 enhances the capacity of AdT to convert Asp–tRNAAsn into Asn–tRNAAsn and Glu–tRNAGln into Gln–tRNAGln but has minimal effect on ND–AspRS function. We discovered that Hp0100 is an ATPase which contains two distinct ATPase active sites that are activated by either of the two misacylated tRNAs, Glu–tRNAGln or Asp–tRNAAsn. The first ATP binding motif shares sequence similarity to adenine nucleotide alpha hydrolase–like (AANH–like) ATP binding motif superfamily. Surprisingly, mutations in this domain only disrupted the Glu–tRNAGln induced ATPase activity; the Asp–tRNAAsn activity was less affected (∼;50% decrease). The second motif shares sequence homology to the P–loop ATP binding motif. In contrast to AANH, P–loop mutations disrupted Asp–tRNAAsn induced ATPase activity but Glu–tRNAGln catalyzed ATPase activity was only partially reduced (∼;50% decrease). These results revealed that there are probably two mutually exclusive ATPase motifs in Hp0100 that are separately activated by Asp–tRNAAsn and Glu–tRNAGln. In addition, our mutagenesis studies also revealed the requirement of each ATPase motif for the corresponding acceleration in the rate of AdT transamidation of either Asp–tRNAAsn or Glu–tRNAGln. Overall, our results highlight the importance of the novel ATPase Hp0100, for the indirect biosynthesis of Asn–tRNAAsn and Gln–tRNAGln in H. pylori.

H. pylori is an obligate human pathogen responsible for causing stomach ulcers and cancer. Its clade (the ε–proteobacteria) includes several human enteric pathogens like Campylobacter jejuni (C. jejuni) that cause other deleterious health problems in humans. Here we describe a unique mechanism used by this clade to ensure accuracy during indirect tRNA aminoacylation. Elucidation of the mechanisms used by other organisms holds potential for the development of a greater understanding of bacterial phylogenetics, speciation, and the identification of novel, clade specific targets for new antibiotics.

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