NMR studies of RNA binding domains of human lysyl aminoacyl tRNA synthetase

<p>Human lysyl aminoacyl tRNA synthetase (hLysRS) is a multi-functional aminoacyl tRNA synthetase which is primarily involved in protein biosynthesis as well as crucial processes ranging from proinflammatory response to signal transduction. One important, non-canonical function of hLysRS is to target lysyl tRNA isoacceptor 3, the HIV-1 reverse transcription primer molecule, for uptake and packaging into new HIV-1 particles. Since the anticodon binding (ACB) domain of hLysRS is required for proper recognition of its cognate tRNA, NMR studies of the ACB domain were conducted to enhance our understanding of how hLysRS interacts with these RNAs during protein biosynthesis as well as HIV-1 viral packaging. The backbone and side chain NMR resonance assignments were obtained for the uniformly 15N-, 13C-labeled ACB domain of hLysRS. With NMR resonance assignments in hand, we mapped the ACB protein surface that is affected by RNA binding via NMR chemical shift perturbation techniques. The RNA molecules studied this way include the anticodon stem-loop of lysyl tRNA isoacceptor 3 (ACSL) and a tRNA-like element (TLE) derived from a region 5’ to the primer binding site of the HIV-1 RNA genome (wild type TLE, or wtTLE). The TLE was recently proposed to compete against lysyl tRNA isoacceptor 3 for interaction with the hLysRS which is important for proper primer binding and reverse transcription during the HIV-1 life cycle. Considerable overlap was observed in terms of the ACB surface affected by RNA binding for the ACSL and wtTLE RNAs, which means the ACB domain interacts with the ACSL and wtTLE in a similar manner. This result supports the hypothesis that the TLE can mimic the tRNA anticodon loop in terms of interacting with the ACB domain of hLysRS.</p><p>An important characteristic of higher eukaryotic forms of lysyl aminoacyl tRNA synthetase (LysRS) is the appendage of an N-terminal domain to the highly conserved ACB and catalytic domains. The function of this N-terminal domain has been proposed to enhance the overall RNA binding affinity which may contribute to the HIV-1 reverse transcription primer uptake and possibly modulate LysRS binding to other proteins; however, its structure still remains unknown. To better elucidate the RNA binding behavior and potential structure of this appendage domain, the backbone and side chain NMR resonance assignments have been achieved for uniformly 15N-, 13C-labeled hLysRS N-terminal domain alone, as well as complexed to ACSL RNA. Based upon the assigned NMR resonances of this protein, the isolated N-terminal domain is mostly unstructured in solution. However, this domain was found to adopt a 27 residue long helical structure only after binding to ACSL RNA. NMR titration studies using different forms of the N-terminal domain protein via segmental labeling reveal that RNA binding by this N-terminal domain is cooperative with the adjacent ACB domain. The fact that the N-terminal domain is unaffected by titration with oligo U or oligo C RNAs regardless of whether the ACB domain is attached suggests that the N-terminal domain may have a preference for structured, possibly stem-loop RNA molecules.</p>

Name thesis:degree_name

PhD

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Arts and Sciences: Chemistry

Grantor dc:publisher

University of Cincinnati

Year dc:date

2012

Author dc:creator

• Liu, Sheng

Contributors dc:contributor

• Tsang, Pearl

• Biophysics
• LysRS
• HIV-1
• anticodon
• N-terminal appendage
• NMR
• lysyl tRNA

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=ucin1353343207

OAI identifier oai:identifier

oai:etd.ohiolink.edu:ucin1353343207