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The T box riboswitch RNA controls the expression of many genes in Gram-positive bacteria, including aminoacyl-tRNA synthetase genes, amino acid biosynthesis genes and amino acid transport genes. By sensing the charge ratio of the cognate tRNA, the T box riboswitch RNA can form two mutually exclusive secondary structures, terminator and antiterminator, to regulate the down stream gene transcription. The uncharged tRNAs bind and stabilize the antiterminator structure through the base pairing of the tRNA accepter end with four bases in the bulge region of the highly conserved T box antiterminator RNA, resulting in complete transcription of the gene. The T box riboswitch RNA is considered to be a possible drug target, since previous studies showed that mutation within this region could reduce bacterial cell growth. Thus, the goal of this research project is to identify ligands that can specifically bind to the T box antiterminator RNA, disrupt tRNA-antiterminator interaction, facilitate the terminator formation and inhibit gene transcription. The ligands tested in this study were oxazolidinone compounds, triazole compounds and amino alcohol compounds. Three high throughput screening assays and one in vitro transcription assay have been successfully developed to identify the lead compounds as potential antibacterial agents. First of all, a fluorescence resonance energy transfer (FRET) assay was refined and used to measure the ligand binding affinity and specificity to the antiterminator RNA model AM1A (wild type) and C11U (reduced-function variant). A high throughput fluorescence anisotropy screening assay was developed and used to identify the T box antiterminator ligands that can effectively disrupt the formation of tRNA-AM1A complex. A high throughput fluorescence-monitored thermal denaturation assay was developed and used to test the ligand effect on the stability of antiterminator model AM1A. Four lead compounds, GHB-7, GHB-54, GHB-134 and WZB-72, were identified and each of them has unique characteristics to interact with the antiterminator RNAs. Finally, a multi-round in vitro transcription assay was optimized and developed for use as a ligand screening assay to test the effect of lead compounds on the T box transcription antitermination. Except that GHB-54 greatly enhanced the antitermination event, the other three lead compounds did not induce significant changes. Both in-line and RNase V1 probing methods were used to characterize the ligand-AM1A interactions. The results indicated that lead compounds GHB-7 and WZB-72 both bind to the lower bulge region of AM1A, however they induce different conformational rearrangements on the A2 helix.