Co-translational Targeting by Signal Recognition Particle Activates Only after Cytosolic Exposure of Signal Sequence

Abstract

Signal recognition particle (SRP) and its receptor (SR) co-translationally target membrane and secretory proteins to membrane specifically and efficiently through multiple layers of regulation. Differential SRP binding to ribosome, SRP-SR association rate, and GTP hydrolysis ensure specificity for substrate over non-substrate. However, there is a debate on whether SRP interacts with its substrate when a target sequence is still buried inside ribosome exit tunnel during the early stage of translation. Previous studies, which only focus on SRP binding to ribosome, produce contradictory results. In this study, we examine the full picture of SRP's multiple layers of regulation at each translation intermediate carrying increasing length of nascent chain. FRET-based SRP-ribosome binding affinity assay and fluorescence detected SRP-SR association rate measurement give the formation of key intermediates in SRP targeting cycle as a function of nascent chain length. Both parameters differ for more than two orders of magnitude between N-terminal and full exposure of targeting sequence from the end of ribosome exit tunnel. Such discrimination is comparable to the difference between substrate and non-substrate. Using e. coli as a model system, our results show that SRP targets its substrate exclusively after the emergence of a transmembrane domain or signal sequence from ribosome exit tunnel.