Abstract
We employ a reporter assay and SHAPE-seq to study translational regulation by RNA-binding proteins, in bacteria. For the reporter assay, we designed 50 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Qβ, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on the position of the hairpin, and exhibits three-nucleotide periodicity. For hairpin positions within the initiation region, in the presence of cognate RNA-binding protein, we observe strong translational repression. In vivo SHAPE-seq results for a representative construct indicate that repression correlates with protection of both the hairpin and the ribosome binding site. Our data suggest that the RBP-hairpin complex entraps the 30S subunit, thereby stalling initiation. We utilize the repression phenomenon in a high-throughput assay for quantitative study of protein-RNA binding in vivo.