Assembly And Function Of Macromolecular Complexes For Accurate Trna Aminoacylation In Helicobacter Pylori

<p> <strong>ABSTRACT</strong> </p> <p> <strong>ASSEMBLY AND FUNCTION OF MACROMOLECULAR COMPLEXES FOR ACCURATE TRNA AMINOACYLATION IN <i>HELICOBACTER PYLORI</i> </strong> </p> <p>by</p> <p> <strong>GAYATHRI SILVA</strong> </p> <p>January 2014</p> <p>Advisor: Dr. Tamara L. Hendrickson</p> <p>Major: Chemistry (Biochemistry)</p> <p>Degree: Doctor of Philosophy</p> <p> <strong>Abstract</strong> </p> <p>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 <i>Helicobacter pylori</i> (<i>H. pylori</i>), 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–tRNA^Asn and Gln–tRNA^Gln. The first step of indirect synthesis of Asn–tRNA^Asn involves misacylation of tRNA^Asn by non–discriminating aspartyl tRNA synthetase (ND–AspRS) to produce Asp–tRNA^Asn. Next, the misacylated tRNA is converted to Asn–tRNA^Asn by Asp–tRNA^Asn/Glu–tRNA^Gln amidotransferase (Asp/Glu–AdT) to produce Asn–tRNA^Asn. Gln–tRNA^Gln is produced vial an analogous process, relying on a misacylating glutamyl tRNA synthetase, GluRS2 and AdT.</p> <p>Including <i>H. pylori</i>, organisms that indirectly synthesize Asn–tRNA^Asn and Gln–tRNA^Gln 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 <i>Thermus thermophilus</i> (<i>T. thermophilus</i>), utilize a tRNA–dependent ribonucleoprotein complex (RNC) called the transamidosome. The <i>T. thermophilus</i> transamidosome contains AdT, tRNA^Asn, and an archaeal ND–AspRS. This Asn–transamidosome traps misacylated Asp–tRNA^Asn until it has been converted to Asn–tRNA^Asn by AdT. Similarly, the thermophilic archaeon <i>Methanothermobacter thermoautotrophicus</i> utilizes a Gln–transamidosome for the synthesis of Gln–tRNA^Gln. This complex consists of ND–GluRS, tRNA^Gln and GatDE. (GatDE is a heterodimeric homolog of AdT.)</p> <p>In contrast to <i>T. thermophilus</i>, <i>H. pylori</i> utilizes a bacterial ND–AspRS that has an extra domain that could sterically prevent transamidosome assembly. <i>H. pylori</i> also requires AdT for conversion of both Asp–tRNA<i>Asn</i> and Glu–tRNA<i>Gln</i> into their cognate aa–tRNAs. In fact the <i>H. pylori</i> Asn– and Gln–transamidosomes were not stably isolated <i>in vitro</i>, suggesting a requirement for an alternative mechanism. </p> <p>We describe the first characterization of a novel protein called Hp0100 in <i>H. pylori</i>, 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–tRNA^Asn into Asn–tRNA^Asn and Glu–tRNA^Gln into Gln–tRNA^Gln 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–tRNA^Gln or Asp–tRNA^Asn. 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–tRNA^Gln induced ATPase activity; the Asp–tRNA^Asn 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–tRNA^Asn induced ATPase activity but Glu–tRNA^Gln 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–tRNA^Asn and Glu–tRNA^Gln. 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–tRNA^Asn or Glu–tRNA^Gln. Overall, our results highlight the importance of the novel ATPase Hp0100, for the indirect biosynthesis of Asn–tRNA^Asn and Gln–tRNAGln in <i>H. pylori</i>. </p> <p> <i>H. pylori</i> is an obligate human pathogen responsible for causing stomach ulcers and cancer. Its clade (the ε–proteobacteria) includes several human enteric pathogens like <i>Campylobacter jejuni</i> (<i>C. jejuni</i>) 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.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Open Access Dissertation

Discipline thesis:degree_discipline

Chemistry

Year dc:date.available

2014

Author dc:creator

• Silva, Gayathri Niroshani

Contributors dc:contributor

• Tamara L. Hendrickson

• Biochemistry
• Chemistry

Repository record dc:identifier

https://digitalcommons.wayne.edu/oa_dissertations/921

OAI identifier oai:identifier

oai:digitalcommons.wayne.edu:oa_dissertations-1920