STUDIES OF THE PYRROLYSYL-TRNA SYNTHETASE

Research in the methylamine metabolish of Methanosarcina spp. led to the discovery of pyrrolysine, the 22nd amino acid encoded by the UAG codon. Pyrrolysyl-tRNA synthetase (PylS) is the specific enzyme that can attach Pyl to its cognate tRNA, the amber suppressor tRNAPyl. The biosynthesis of pyrrolysine required the function of three genes, pylC, pylB, pylD, which were identified in the same apparent transcriptional unit as pylS and pylT. Expression of all five archaeal pyl genes will allow E. coli cells to translate UAG as pyrrolysine. A recent search on the completed genome database on NCBI revealed nine Archaeal and nine Bacterial genomes to contain the pyl genes. The archaeal pylS gene appears to be represented by two separated genes in bacteria, pylSc and pylSn. The gene product of pylSc is homologous to the C-terminus of PylS and contains the three conserved motifs of class II aaRS. The gene product of pylSn is homolog to the N-terminus of PylS and contains a high percentage of positive charged residues. Gram positive bacterium Desulfitobacterium hafniense is the first bacterial organism to be identified to contain pyl genes. The activity of D. hafniense PylSc was examined here and the results showed that this protein possesses in vivo and in vitro aminoacylation activity. However, Electrophoretic Mobility Shift Assays (EMSA) indicated that PylSc does not possess as high affinity for tRNAPyl as PylS does. These results suggested that the C-terminus of PylS contains a pyrrolysyl-tRNA synthetase catalytic domain while the N-terminus is responsible for the high affinity for tRNAPyl (chapter 2). Results presented in chapter 3 showed that D. hafniense PylSn binds tRNAPyl specifically with high affinity. Substitutions of conserved residues in tRNAPyl in the variable loop, D-stem, and T stem & loop had significant impact in PylSn binding. PylSn and the N-terminus of PylS share no sequence similarity to any protein with known function and comprise the protein superfamily TIGR03129. The presence of this protein superfamily is limited to organisms that contain genes for the biosynthesis of pyrrolysine. These results suggest that the N-terminus of PylS as well as PylSn represent a novel tRNAPyl binding domain (chapter 3). The specificity constant of PylS for pyrrolysine is much higher than other analogs. Part of this specificity lies within the methylpyrroline ring of pyrrolysine. The data presented in chapter 4 showed that the activation of pyrrolysine but not 2-Thf-lys, an analog which also contains electronegative oxygen at the corresponding position of imine nitrogen, by PylS is significantly inhibited under higher pH values (pH 7.0 – pH 8.8). The slow activation of pyrrolysine by PylS under higher pH values is apparently due to increased Km as indicated by apparent kinetic parameters. The results indicated the ionizable imine nitrogen is critical for PylS recognition of pyrrolysine, presumabably by forming a charged hydrogen bond with PylS. A strictly conserved Tyr384 has been proposed to hydrogen-bonding with pyrrolysine imine nitrogen. Here, a Y384F mutation on PylS (PylSYF) was shown to dramatically impair the specificity of PylS for pyrrolysine in ATP:PPi exchange assays. Together, this data suggested the ionization of pyrrolysine imine nitrogen in amino acid substrate specificity of PylS. The role of Tyr384 in pyrrolysine activation was also indicated in this dissertation. (chapter 4).

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biochemistry Program, Ohio State

Grantor dc:publisher

The Ohio State University

Year dc:date

2013

Author dc:creator

• Jiang, Ruisheng

Contributors dc:contributor

• krzycki, joseph

• Biochemistry
• Microbiology
• Archaeology
• pyrrolysine
• Amino Acid
• Aminoacyl tRNA Synthetase
• Archaea
• Bacteria
• RNA-binding Protein
• Transfer RNA (tRNA)
• 22nd Amino Acid
• Genetic Code
• Pyrrolysyl-tRNA Synthetase

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=osu1365512882

OAI identifier oai:identifier

oai:etd.ohiolink.edu:osu1365512882