Interaction between Transcription and Translation Machineries on a Nascent RNA with Higher Order Structure

Abstract

During bacterial transcription, pausing of the RNA polymerase (RNAP) is an important feature that allows co-transcriptional RNA folding as well as protein recruitment. It has long been proposed that RNAP pausing in the vicinity of the translation start site allows the ribosome to bind the messenger RNA (mRNA) in order to couple the translation with the neo-synthesis of the transcript. There is a growing interest to understand the physical interaction between the ribosome and the RNAP in elongation complex (EC). Here, using both biochemical and single molecule fluorescence assays, we have investigated the mechanism of transcription-translation coupling of a pseudoknot-structured class 1 preQ1 riboswitch with higher order pseudoknot structure. Our results demonstrate that the RNAP significantly stabilizes the initial binding of the ribosome on the nascent riboswitch transcript. In addition, while using the preQ1 ligand that binds to the riboswitch with high affinity to form a folded conformation of the pseudoknot, we show that the conformational stability of the nascent RNA also plays important role for initiating the ribosome binding. Furthermore, our results indicate that transcriptional factor NusG, which is known to establish a bridge between the RNAP and the ribosome, stabilizes ribosome binding on the nascent RNA. Our transcription assays show that, interaction with the ribosome significantly stabilizes the RNAP pause at close to the translation start site. However, while translating, the ribosome releases the stalled RNAP showing that transcription-translation coupling is determinant for transcription progression. Currently we are working on a protein induced fluorescence enhancement (PIFE) assay to understand the effect of the rate of translation onto the rate of transcription by RNAP. These results provide significant insights to understand the cooperative mechanism of transcription and translation, and its effect on overall gene expression.