Quantifying the Spatial Organization of Bacterial Ribosomes using Three-Dimensional Super-Resolution Microscopy

Abstract

Correctly localizing proteins is essential for many biological processes. Since bacteria lack organelles compartmentalizing transcription and translation, it is possible that the site of protein synthesis is organized around the location of the corresponding gene. This would provide a mechanism to create a high local concentration of a specific protein. Past work supports this hypothesis of complete spatial and temporal overlap between the positions of active ribosomes and the corresponding gene (model 1). However, other results support a conflicting model with separation (on the order of hundreds of nanometers) between the gene and its site of translation (model 2). The latter case also implies that transcription and translation do not occur simultaneously. The small size of bacteria prohibits using conventional fluorescence imaging to determine the positions of individual ribosomes. To overcome this, we apply the method of 3D super-resolution microscopy which reveals individual ribosome position with tens of nanometer precision. In order to distinguish between models 1 and 2, we combine 3D super-resolution microscopy with the translation level of individual genes mapped to cell position on a population of Caulobacter crescentus cells in the same developmental state. According to model 1, high spatial density of ribosomes correlates with the positions of highly translated genes on the chromosome. We quantify the level of correlation for individual cells and compare the correlation with simulated ribosome positions from model probability distributions. Combining high resolution single molecule information with translation profiling data reveals the underlying spatial heterogeneity of ribosome organization on a well-defined system.