Development of cysteine-specific tRNAs for site-specific protein fluorescence labeling and co-translational protein folding studies
Date Issued
2014
Date
2014
Author(s)
Koubek, Ji??
Abstract
Ensuring labeling specificity for fluorophore labeling is a challenge in fluorescent spectroscopy. This biophysical technique is one of the primary research tools for studying cotranslational protein folding which studies protein conformation before it has been released from the ribosome. Due to the nature of ribosome-bound nascent chains (RNCs), fluorescent labeling must be coupled with translation during which tRNA acts as the carrier of fluorescent amino acid. In this work, a novel overexpressed suppressor tRNAcysAmber is developed for the production of BODIPY FL-labeled RNCs. In vitro transcription as well as overexpression is tested as the methods of suppressor tRNA production. In order to simplify the purification procedures, Bacillus subtilis tRNAcysAmber has been selected for its distinctive sequence from any endogenous E. coli RNA. In a single purification step, ample amounts of tRNAcysAmber have been obtained. As cysteinyl-tRNA synthetase was previously shown to aminoacylate tRNAcysAmber with low efficiency, several point mutations were introduced into the C-terminus of cysteinyl-tRNA synthetase to compensate for the Amber mutation in the tRNA anticodon loop. Out of the cysteinyl-tRNA synthetase mutants, D436S mutant is shown with improved aminoacylation efficiency and specificity towards tRNAcysAmber. In addition, overexpressed BODIPY FL-cysteinyl-tRNAcysAmber indicates improved stability of this tRNA compared to the in vitro transcribed tRNA. Applying this tRNA, the dynamics of single-labeled RNCs by time-resolved anisotropy was studied to reveal information about the protein folding on the ribosome. The natively unfolded phosphorylated insulin receptor domain (PIR) and the zinc-induced folding in zinc-finger RNC help to correlate the fluorescence correlation time with different nascent chain movements. To further study the impact of chaperones on the RNC dynamics at different stages of translation, Entner-Douderoff aldolase (Eda) RNCs with four predetermined chain length are generated in either the wild-type or chaperone-depleted cell-free system. By applying BODIPY FL-cysteinyl-tRNAcysAmber, our results indicate that Eda may start folding without chaperones after approximately half of the protein emerges from the ribosome. In addition, chaperones increase the nascent chain confinement in the full length Eda RNC, which may cause the decrease the binding of the trigger factor, a co-translational chaperone, due to growing hindrances between RNCs and chaperones. Overall, the facile preparation of suppressor tRNA for labeling with fluorophores is demonstrated together with the application of single-residue labeled nascent chains in studying the effect of chaperones on the RNC dynamics.
Subjects
Protein labeling
tRNA
time-resolved fluorescence anisotropy
co-translational protein folding
chaperones
in vitro translation
RNA purification
Type
thesis
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