Fig. 1

Elements, utility, and complete protocol of INTERFACE. a Moving from 5′ to 3′, each INTERFACE is composed of a unique asRNA probe, followed by the RSE, then the ES, comprised of tnaC and the rut site, and then the RER (a truncated gfp gene). INTERFACE can adopt two distinct RNA conformations depending on asRNA probe:taRNA region binding that determines the exposure of the RBS upstream of the ES: (i) a structured hairpin in which the RBS is occluded and (ii) an unstructured domain in which the RBS is exposed. b When targeting an accessible region (left panel), the INTERFACE asRNA probe ‘X’ binds strongly, releasing the RBS for tnaC translation. Translation of tnaC causes ribosomal stalling near the rut. The stalled ribosome physically occludes the Rho factor from the rut site which enables transcriptional elongation. In contrast, INTERFACE targeting of an inaccessible RNA region with asRNA probe ‘Y’ (right panel) generates a truncated transcript. Specifically, the absence of tnaC expression due to lack of binding between the asRNA probe and the taRNA enables rut site availability for Rho-dependent transcriptional termination. c (i) A library of asRNA probes is inserted into either of two parent plasmids (Supplementary Fig. 1). (ii) The library is transformed into the relevant experimental strain as described in Methods section. (iii) The INTERFACE system is induced and the library of asRNA probes interact with corresponding taRNA regions in vivo to varying degrees, generating a spectrum of transcript lengths bar-coded with the probe sequence at the 5′-end. (iv) Total RNA, including transcripts of interest containing corresponding probes (green and orange 5′-ends circled in red), is extracted. (v) A cDNA library for Illumina-platform RNA-seq is generated, in which INTERFACE RNA is among the pool of adapter-ligated RNA and consequently in the final cDNA library (red). (vi) RNA-seq is performed per standard procedures in a paired-end 75 × 2 run in the Illumina platform. (vii) Mapping against synthetic INTERFACE library is performed to exclude non-INTERFACE transcripts and associate corresponding 3′-ends (R2) to 5′-ends (R1) to determine the length of each region-specific INTERFACE transcript