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We employ a reporter assay and Selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 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 hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wideswath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation.Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo. INTRODUCTIONThe regulation of gene expression is a process central to all biological life-forms. It is a process thought to be mediated largely by proteins, which interact with either chromatin or its RNA product.The best-known form of regulation is mediated by transcription factors, which control RNA levels by their sequence-specific interaction with DNA. Post transcriptional regulation based on protein-RNA interactions, however, is quite different, due to the nature of RNA. Unlike DNA which is a long, chromatinized, replicated, and for the most part exists as a double stranded molecule, RNA is a short, transient (i.e. constantly manufactured and degraded), exists in multiple copies, has particular modifications (1, 2), and folds into functional secondary and tertiary structures. RNA structure is

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We employ a reporter assay and Selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 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 hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wideswath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation.Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo. thought to be highly dynamic, and is dependent on many factors such as temperature, cellular RNAbinding protein (RBP) content, presence or absence of translating ribosomes, and interaction with other RNA molecules (3). Thus, a typical RBP-RNA interaction is likely to depend not only on the presence of a specific binding sequence, but also on many other factors.In bacteria, post-transcriptional regulation has been studied extensively in recent decades.There are well-documented examples of RBPs that either inhibit or directly compete with ribosome binding via a variety of mechanisms. These include direct competition with the 30S ribosomal subunit for binding via single stranded recognition (4), entrapment of the 30S subunit in an inactive complex via a nested pseudoknot structure (5) and ribosome assembly inhibition when the RBP is bound to a structured RBP binding site, or hairpin (6-9). These hairpins have been studied in three distinct positions: either immediately downstream and including the AUG (7), upstream of the Shine-Dalgarno sequence (8), or as structures that entrap Shine-Dalgarno motifs, as is the case for the PP7 and MS2 phage coat-protein binding sites. There is also a well-characterized example of translation stimulation:binding of the phage Com RBP was shown to destabilize a sequestered ribosome binding site (RBS) of the Mu phage mom gene, thereby facilitating translation (10, 11). While these studies indicate a richness of RBP-RNA-based regulatory mechanisms, a systematic understanding of the relationship between RBP binding, sequence specificity, the underlying secondary and tertiary RNA structure, and the resultant regulatory output is still lacking.In recent years, advances in next generation ...

We explore a model for ‘quenching-like' repression by studying synthetic bacterial enhancers, each characterized by a different binding site architecture. To do so, we take a three-pronged approach: first, we compute the probability that a protein-bound dsDNA molecule will loop. Second, we use hundreds of synthetic enhancers to test the model's predictions in bacteria. Finally, we verify the mechanism bioinformatically in native genomes. Here we show that excluded volume effects generated by DNA-bound proteins can generate substantial quenching. Moreover, the type and extent of the regulatory effect depend strongly on the relative arrangement of the binding sites. The implications of these results are that enhancers should be insensitive to 10–11 bp insertions or deletions (INDELs) and sensitive to 5–6 bp INDELs. We test this prediction on 61 σ54-regulated qrr genes from the Vibrio genus and confirm the tolerance of these enhancers' sequences to the DNA's helical repeat.