Patent ID: 7413856
Filing Date: 2008-08-19
Classification: C12Q

Abstract:
1. A method for screening substances which are potential inhibitors of expression of bacterial T-box regulated genes, comprising the steps of: a) incubating one or more assay mixtures to produce a readthrough mRNA product, wherein the assay mixtures comprise: a template DNA that comprises: (i) a bacterial promoter, (ii) a glyQS leader, including a transcription start site, and (iii) a downstream polynucleotide of sufficient length for detection of a read-through mRNA product; divalent magnesium cations; nucleoside triphosphates; dinucleotides corresponding to and encoded by the transcription start site of the glyQS leader; bacterial RNA polymerase complex; and uncharged tRNA specific for a specifier sequence located in the glyQS leader; and b) incubating a potential inhibitor substance with one or more assay mixtures to produce a readthrough mRNA product, wherein the assay mixtures comprise: a template DNA that comprises: (i) a bacterial promoter, (ii) a glyQS leader, including a transcription start site, and (iii) a downstream polynucleotide of sufficient length for detection of a read-through mRNA product; divalent magnesium cations; nucleoside triphosphates; dinucleotides corresponding to and encoded by the transcription start site of the glyQS leader; bacterial RNA polymerase complex; and uncharged tRNA specific for a specifier sequence located in the glyQS leader; c) comparing the fraction of total mRNA products corresponding to the read-through mRNA product produced in step a) with the fraction of total mRNA products corresponding to the read-through mRNA product produced in step b) wherein a lesser fraction of the read-through mRNA product produced in step b) in comparison with step a) indicates that said potential inhibitor substance inhibits transcriptional readthrough of said glyQS leader and therefore is an inhibitor of expression of bacterial T-box regulated genes.