RNA-binding proteins (RBPs) control the fate of nearly every transcript in a 
cell. However, no existing approach for studying these posttranscriptional gene 
regulators combines transcriptome-wide throughput and biophysical precision. 
Here, we describe an assay that accomplishes this. Using commonly available 
hardware, we built a customizable, open-source platform that leverages the 
inherent throughput of Illumina technology for direct biophysical measurements. 
We used the platform to quantitatively measure the binding affinity of the 
prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae 
genome. The scale and precision of these measurements revealed many previously 
unknown features of this well-studied RBP. Our transcribed genome array (TGA) 
assayed both rare and abundant transcripts with equivalent proficiency, 
revealing hundreds of low-abundance targets missed by previous approaches. These 
targets regulated diverse biological processes including nutrient sensing and 
the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA 
provided single-nucleotide resolution for each binding site and delineated a 
highly specific sequence and structure motif for Vts1 binding. Changes in 
transcript levels in vts1Δ cells established the regulatory function of these 
binding sites. The impact of Vts1 on transcript abundance was largely 
independent of where it bound within an mRNA, challenging prevailing assumptions 
about how this RBP drives RNA degradation. TGA thus enables a quantitative 
description of the relationship between variant RNA structures, affinity, and in 
vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will 
provide similarly comprehensive and quantitative insights into the function of 
virtually any RBP.