Abstract
Transfer RNA (tRNA) links messenger RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite the importance of tRNA for translation, its subcellular distribution and diffusion properties in live cells are poorly understood. Here, we provide the first direct report on tRNA diffusion localization in live bacteria. We internalized tRNA labeled with organic fluorophores into live bacteria, applied single-molecule fluorescence imaging with single-particle tracking and localized and tracked single tRNA molecules over seconds. We observed two diffusive species: fast (with a diffusion coefficient of ∼8 μm2/s, consistent with free tRNA) and slow (consistent with tRNA bound to larger complexes). Our data indicate that a large fraction of internalized fluorescent tRNA (>70%) appears to diffuse freely in the bacterial cell. We also obtained the subcellular distribution of fast and slow diffusing tRNA molecules in multiple cells by normalizing for cell morphology. While fast diffusing tRNA is not excluded from the bacterial nucleoid, slow diffusing tRNA is localized to the cell periphery (showing a 30% enrichment versus a uniform distribution), similar to non-uniform localizations previously observed for mRNA and ribosomes.
Highlights
Studying the subcellular distribution of RNA in living cells is vital for understanding the spatial organization of gene expression [1], since this distribution can control the processes of transcription and translation and efficiently alter protein activity
A preferential localization of ASH1 mRNA encoding for an unstable transcriptional repressor protein in daughter cells has been shown in budding yeast [3], and translation-independent subcellular localization of transcripts encoding for membrane and soluble proteins has been observed in bacteria [4]
Organic dyelabeled bulk E. coli Transfer RNA (tRNA) molecules (Figure 1A), as well as RNA25 were electroporated into electrocompetent E. coli DH5␣ cells (‘Materials and Methods’ section) and imaged under highly-inclined and laminated optical sheet illumination [40] in the respective fluorescent channel (Figure 1B)
Summary
Studying the subcellular distribution of RNA in living cells is vital for understanding the spatial organization of gene expression [1], since this distribution can control the processes of transcription and translation and efficiently alter protein activity. FPs are less bright and photostable than organic fluorophores [8,9] and require more than 24 copies of MS2 binding sites (accommodating 48 GFPs) to localize single mRNA molecules [10], making the MS2 array a bulky label (200 × 5 × 3 nm) that might perturb mRNA interactions. An alternative approach used live-cell tracking of mRNA molecules singly labeled using organic fluorophores, e.g. by tagging the MS2 coat protein via a polypeptide linker (SNAP tags [11]), or through RNA aptamers [12,13]; the latter method has been used for tracking single mRNA molecules in mammalian cells [14]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
