Doctor of Philosophy (PhD), Ohio University, 2009, Chemistry and Biochemistry (Arts and Sciences)

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 Mg2+ 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 (open full item for complete abstract)

Committee: Jennifer Hines PhD (Advisor); Mark McMills PhD (Committee Member); Shiyong Wu PhD (Committee Member); Soichi Tanda (Committee Member) Subjects: Chemistry