Methodology for Ribosome Profiling of Key Stages of the Caulobacter crescentus Cell Cycle

Abstract

Bacterial cell division is the result of a productive round of the cell cycle to yield two daughter cells. The cell cycle is highly coordinated in Caulobacter crescentus where it is driven by a cell cycle gene-regulatory network that coordinates gene expression with the major cell cycle events such as chromosome replication and cell division. Recent ribosomes profiling data showed that 484 genes undergo changes in translation efficiency during the cell cycle, suggesting a broad role for translational control in cell cycle regulation. In this chapter, we focus on how to perform ribosome profiling to measure the translation efficiency across cellular mRNAs at key stages in the Caulobacter cell cycle. This methodology relies on the high-yield ludox gradient synchronization of Caulobacter cells followed by ribosome profiling to measure ribosome density and total RNA-seq to measure mRNA levels.

Keywords: Caulobacter crescentus; Cell cycle; Ribosome profiling; mRNA translation.

Figure 1

Schematic of Caulobacter ribosome profiling procedure. Section 2.1 - Cell Growth, Synchronization and Harvesting: Grow 1 Liter of NA1000 cells, synchronize, and then grow to one of the cell cycle time points. Section 2.2 - Harvesting Cells: To each cell population, harvest by chloramphenicol pre-treatment (2 min at 28 °C) followed by rapid cell collection on ice. Section 2.3 - Cell Lysis: Lysis is performed under liquid nitrogen and a small portion is saved as a frozen aliquot for total RNA-seq. Section 2.4 - Footprinting and Footprint Extraction: Footprint mRNA fragments, select for size, and recover digested mRNA fragments. Section 2.5 - Total RNA-Seq Sample Preparation and PAGE Size Selection: Use the lysate aliquot from section 2.3 and prepare for total RNA-seq. Section 2.6 - Library Generation: Sequencing libraries are generated in the same way from the products of both ribosome footprinting and total RNA-Seq.

Figure 2

Example of Caulobacter cell cycle polysomes. Undigested cell lysate derived from a single cell cycle timepoint was separated on a 10–55% sucrose gradient.

Figure 3

rRNA depletion of total RNA samples for total RNA-seq. Bioanalyzer traces before (top) and after rRNA removal (bottom). Stable RNA peaks are labeled. Caulobacter 23s rRNA is processed into two fragments, one runs at the same size as the 16S rRNA and the other runs at a shorter length. Y-axis scales are different for the two graphs.