Effects of metal ions and loop stability on the structure and function of the T box antiterminator RNA and its complex with model tRNA

The T box antitermination mechanism is a novel transcription regulatory system found in many Gram-positive bacteria. The mechanism serves to regulate aminoacyl tRNA synthetases, amino acid transport genes and amino acid biosynthesis genes. The genes regulated by this system are characterized by highly conserved primary and secondary structural elements in the 5' untranslated region. Two mutually exclusive secondary structures can form in the 5' untranslated region; the antiterminator, stabilized by uncharged tRNA binding, leads to transcription read through and full synthesis of the gene. The formation of the terminator element, in the absence of uncharged tRNA leads to transcription termination. The antiterminator consists of two helices, A1 and A2 separated by a seven-nucleotide bulge and a closing loop in the A2 helix.;This study investigated the effects of the loop region and Mg 2+ on the structure and functions of the antiterminator and microhelix tRNA model. The use of computational, spectroscopic and molecular biology techniques demonstrated that the loop region had an effect on the structure and stability of the antiterminator and microhelix RNA but did not change the overall functions of the antiterminator model.;A series of antiterminator models and microhelix RNA models were constructed with a substitution of well-characterized stable GNRA and UNCG tetraloops. A part from these mutations, the wild type antiterminators AM2 with a loop sequence AAUCA and the GlyQS (Glycine synthetase) with a loop sequence (GAAC) that is not a classified stable loop were also investigated. Using Mfold thermodynamic stability and UV monitored thermal denaturation, it was demonstrated that the loop region contributes significantly to the stability of the antiterminator. There was no clear correlation in the Circular Dichroism (CD) study of the different antiterminators. Enzymatic probing study showed that the loop region affected the A2 helix of the antiterminators but not the nature of the bulge.;Binding studies using fluorescence-based binding assays demonstrated that divalent metal requirement for tRNA binding to the antiterminator RNA is dependent on antiterminator sequence and structure. Monovalent metal ions alone did not sufficiently facilitate tRNA binding to the antiterminator. Interaction of small molecules with different antiterminators showed that the small molecules bind antiterminators differently due to RNA structural differences. Finally this study provided further evidence that binding of tRNA to antiterminator model occurs by means of an induced fit.