Real-Time Messenger RNA Dynamics in Bacillus subtilis

Abstract

Messenger RNA molecules have been localized to different positions in cells and have been followed by time-lapse microscopy. We have used MS2-mVenus–labeled mRNA and single-particle tracking to obtain information on the dynamics of single-mRNA molecules in real time. Using single-molecule tracking, we show that several mRNA molecules visualized via two MS2-binding sites and MS2-mVenus expressed in Bacillus subtilis cells show free diffusion through the entire cell and constrained motion predominantly close to the cell membrane and at the polar regions of the cells. Because constrained motion of mRNAs likely reflects molecules complexed with ribosomes, our data support the idea that translation occurs at sites surrounding the nucleoids. Squared displacement analyses show the existence of at least two distinct populations of molecules with different diffusion constants or possibly of three populations, for example, freely mobile mRNAs, mRNAs in transition complexes, or in complex with polysomes. Diffusion constants between differently sized mRNAs did not differ dramatically and were much lower than that of cytosolic proteins. These data agree with the large size of mRNA molecules and suggest that, within the viscous cytoplasm, size variations do not translate into mobility differences. However, at observed diffusion constants, mRNA molecules would be able to reach all positions within cells in a frame of seconds. We did not observe strong differences in the location of confined motion for mRNAs encoding mostly soluble or membrane proteins, indicating that there is no strong bias for localization of membrane protein-encoding transcripts for the cell membrane.

Real-time dynamics of individual chemoreceptor mRNA molecules reveals translation hotspots at the inner membrane ofEscherichia coli

“…The results of this fitting procedure and all fits to other mRNA variants are presented in Figure 2 and Supplementary Figure 2 and Supplementary Figure 3 . This is in good agreement with a recent finding that mRNA diffusion in Bacillus subtilis shows up to three diffusive states (21), although our bacteria of choice, labelling system and imaging conditions were overall different. A construct with only 6 MBS elements, tsr-6xms2 , showed a nearly identical distribution of subpopulations ( Figure 2A ).…”

“…With few exceptions (21, 22), the majority of recent studies is limited by the choice of experimental methods that do not capture the dynamics of individual mRNA molecules and cannot distinguish between actively translated and unbound mRNA. Here, we study the diffusive dynamics and the spatial distribution of individual mRNA molecules in living Escherichia coli cells using the serine chemoreceptor Tsr as an example transmembrane protein that is translated directly into the membrane by the Sec translocation machinery (23).…”

Protein secretion zones within the Gram-positive cell wall

“…When tracks were sorted into different populations, and tracks of the slow mobile “static” fraction were projected into a medium-sized cell of 3 × 1 µm, most molecules were found close to the cell poles, or in the cell centre, which is very similar to the localization of translating ribosomes 50,54,55 (Fig. 8C).…”

Protein secretion zones within the Gram-positive cell wall

“…When tracks were sorted into different populations, and tracks of the slow mobile "static" fraction were projected into a medium-sized cell of 3 x 1 µm, most molecules were found close to the cell poles, or in the cell centre, which is very similar to the localization of translating ribosomes 50,54,55 (Fig. 8C).…”