Fluorescence and NMR Characterization of a T Box Antiterminator-tRNA Complex

 
 
 
 

Fluorescence and NMR Characterization of a T Box Antiterminator-tRNA Complex

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dc.contributor.advisor Hines, Jennifer V. en_US
dc.contributor.author Means, John A. en_US
dc.date.accessioned 2008-07-08T17:05:24Z
dc.date.available 2008-07-08T17:05:24Z
dc.date.created 2007 en_US
dc.date.issued 2008-07-08T17:05:24Z
dc.identifier.uri http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1194218959 en_US
dc.identifier.uri http://hdl.handle.net/2374.OX/13119
dc.description The T box transcription antitermination system is present in many amino acid biosynthesis, metabolism, and transport, and tRNA synthetase genes in Gram-positive bacteria. The regulatory system involves a novel RNA/RNA interaction in which a complex set of conserved structural and sequence elements within the 5' untranslated leader region of the regulated gene transcript interacts with the uncharged form of the gene's cognate tRNA. As a part of the control mechanism for transcription of the mRNA, two mutually exclusive structures are formed within the 5' leader region – the terminator or antiterminator structure. The cognate tRNA binds to the specifier sequence in stem I of the leader region, but the antiterminator is only stabilized by uncharged cognate tRNA. If a charged tRNA is bound to the specifier sequence, the antiterminator is not stabilized and the terminator forms. Transcription read-through is allowed upon the formation of the antiterminator structure, which consists of a highly conserved seven-nucleotide bulge in which the first four bases of the bulge base pair with the 3' acceptor end of the tRNA. The current studies use models of the Bacillus subtilistyrosyl-tRNA synthetase gene ( tyrS) as a model system for fluorescence and NMR studies of the tRNA acceptor end interaction with the antiterminator. The fluorescence studies have shown that the core fold of the tRNA is necessary for tight binding of the tRNA with the antiterminator. Additional fluorescence studies have shown that the binding of the tRNA with the antiterminator is a two-step process, in which a small conformational change is induced by the first binding step and significant structural reorganization accompanies the second binding step. For this binding interaction to occur, a threshold level of Mg ^2+is necessary for "cationic mediation" of the negatively-charged electrostatic repulsion that arises as the 5'-monophosphate at the end of the tRNA comes into close proximity with the phosphate backbone of the antiterminator bulge. NMR studies have shown that the antiterminator structural dynamics change in the presence of increased Mg ^2+. In addition, the NMR studies have also provided direct evidence of the base pairing between the four tRNA acceptor end nucleotides and their four complementary nucleotides at the 5' end of the antiterminator bulge. Binding-induced structural changes in the antiterminator model and the tRNA model were also detected by the NMR. All of these NMR studies culminated in a low-resolution solution structure of the AM1A/mh-UCCA complex. The fluorescence and NMR studies provide evidence for binding via an induced fit accompanied by structural changes that may act to "lock" the tRNA into its complex with the 5'-untranslated region of the mRNA. en_US
dc.format application/pdf en_US
dc.format 210p. en_US
dc.rights restricted; full text not available online en_US
dc.rights Copyright and permissions information available at the source archive en_US
dc.subject T box antiterminator system en_US
dc.subject mRNA-tRNA interaction en_US
dc.subject 2-aminopurine steady-state fluorescence en_US
dc.subject 2-aminopurine time-resolved fluorescence en_US
dc.subject NMR en_US
dc.title Fluorescence and NMR Characterization of a T Box Antiterminator-tRNA Complex en_US
dc.type Electronic Thesis or Dissertation en_US
dc.degree.name PhD en_US
dc.degree.level doctoral en_US
dc.degree.discipline Chemistry (Arts and Sciences) en_US
dc.degree.grantor Ohio University en_US
dc.contributor.publisher Ohio University / OhioLINK en_US

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