Studies of the 5' Untranslated Region of sagA/Pel in Streptococcus pyogenes.

Streptococcus pyogenes is a human pathogen that is also known as group A Streptococcus (GAS), which is responsible for the development of sore throats and skin infections as well as the more severe condition, necrotizing fasciitis. The streptolysin associated gene A, sagA, codes for the protein streptolysin A (SagA), which is modified and exported as the cytotoxin streptolysin S (SLS). SLS is a virulence factor that inhibits the response of neutrophils during necrotizing fasciitis. The sagA gene is contained within the pleiotropic effect locus (pel). Previous studies have identified sagA as both the gene for the streptolysin S peptide, and the small regulatory RNA (sRNA), known as Pel, which influences expression of multiple virulence genes. The 5' UTR of sagA/Pel contains the regions necessary for both transcription initiation and ribosomal binding for translation. Focusing on the 5' UTR of sagA, we hypothesize that the RNA structure of this region affects the transcription of sagA and the initiation of translation. Our goal is to characterize the secondary and 3D structure of the sagA/Pel mRNA/sRNA, which will allow for a better understanding for the role it plays in streptococcal pathogenesis. In this study, RNA constructs containing predicted structured regions were produced by in vitro transcription. Secondary structures were predicted using the mfold and RNAfold webserver based programs. The predicted secondary structures were used to generate three-dimensional models with the program FARFAR through the ROSIE webserver. Differential scanning fluorimetry was also used to assess the structural stability of the RNA constructs and to screen a variety of conditions to be used in crystallization studies. These experiments provided evidence that the 5' UTR of sagA/Pel contains significant structured regions and optimal conditions were identified for future structural studies. Potential interactions between the sagA/Pel sRNA and regulated mRNA targets were predicted using the TargetRNA2 webserver. The potential sites for recognition were used to evaluate the functional significance of the structured regions within the 5' UTR of sagA/Pel. Identification of structural motifs necessary for regulating the expression of virulence genes will aid in the design of therapeutic strategies to inhibit the production of the virulence factors, such as SLS. In future studies, we plan to use structural techniques such as NMR spectroscopy and X-ray crystallography to help further understand the role that sagA/Pel plays in streptococcal diseases.