AlphaFold 3-powered discovery of phage proteins that inhibit bacterial transcription

Phages are the most abundant biological entities on Earth and play central roles in bacterial evolution and the emergence of new pathogens. Many phages encode proteins that specifically inhibit host RNA polymerase activity, thereby sabotaging and, in some cases, hijacking the host transcription machinery to serve their needs. Identification and characterization of these transcription inhibitors not only provide insights into the logic of transcription regulation but also inspire the design of antibiotics targeting bacterial transcription. Traditional methods for identifying new phage proteins that inhibit bacterial transcription are labor-intensive and require access to live phages. To overcome these limitations, we developed a highly efficient pipeline for AlphaFold 3-guided discovery of phage proteins that inhibit bacterial transcription. Using this pipeline, three phage proteins were identified and characterized. Structural and biochemical analyses demonstrated that these phage proteins bind to distinct sites on RNA polymerase and inhibit transcription via unprecedented mechanisms. This study showcases the power of AlphaFold 3 in discovering novel binders of large protein complexes, and the pipeline developed here could be readily adapted to screen modulators of other large targets, such as the ribosome, proteasome, and CRISPR-Cas systems.