ARN et microfluidique : analyse cinétique du repliement des ARN dans la régulation – RNAµfluidics

RNA, which has long been viewed as a 'passive' information carrier, is now widely recognized as a major actor for the regulation of gene expression. In bacteria, the mRNA is a key player in this process, and the regulation involves mRNA structural elements that directly sense the environmental cues, or facilitate the interaction with either metabolite, target RNA, or RNA-binding proteins. In all these systems, regulation is highly dependent on RNA structural switches that take place during the transcription process. In this project we will study such mechanisms, and embark on the kinetic aspects in relation with mRNA folding. Experimentally, we will use well-mastered probing techniques in solution but in the frame of microfluidics, which offers a large array of new possibilities and advantages in comparison of usual quenched-flow or stopped-flow systems. Among them are miniaturization allowing to use less biological material (and also less harmful chemicals), as well as to increase the complexity and versatility of the kinetic lines. We will consider two bacterial systems, first the Escherichia coli thiamine pyrophosphate (TPP) riboswitch which controls translation initiation of thiM gene and, second, the Staphylococcus aureus RNAIII which regulates the translation of many different mRNAs encoding virulence factors. RNAIII is responsible, inter alia, for the inhibition of translation of spa mRNA by sequestrating its ribosome binding site, and for the activation of translation of hla mRNA by inducing a mRNA structural switch that facilitates ribosome binding. We want to analyse the kinetics of sequestration from the first recognition event (formation of a loop-loop complex) to full duplex formation between RNAIII and spa mRNA. The influence of the RNA chaperone Hfq protein will be studied on hla mRNA translation activation mediated by RNAIII. The TPP riboswitch, whose crystal structure is known and which has already been studied kinetically, will be used in a first step as a model system to allow us assessing the microfluidic technology that will be developed. Such developments will allow us addressing more complex questions about the kinetic aspect of RNA-mediated regulation and will participate in making the extremely promising microfluidic technology available at the bench level of biochemical laboratories.