Bacteria have a complex internal organization with specific localization of many proteins and DNA, which dynamically move during the cell cycle and in response to changing environmental stimuli. Much less is known, however, about the localization and movements of RNA molecules. By modifying our previous RNA labeling system, we monitor the expression and localization of a model RNA transcript in live Escherichia coli cells. Our results reveal that the target RNA is not evenly distributed within the cell and localizes laterally along the long cell axis, in a pattern suggesting the existence of ordered helical RNA structures reminiscent of known bacterial cytoskeletal cellular elements.espite their relatively small dimensions, bacterial cells show a remarkable, rich internal subcellular organization that has captured the interest of researchers over the past decade (1-4). Many cytoplasmic and membrane proteins, particularly those involved in cell division, DNA replication, and chromosome segregation, have specific subcellular localizations that can change quickly over time in response to cell cycle progression, motility, and environmental cues. This dynamic and organized behavior is also true for bacterial chromosomal DNA. The use of GFP fusions and in situ fluorescence hybridization (FISH) have shown that every chromosomal locus has a defined subcellular address and is replicated and segregated into the new cell as part of an active and directed process (4, 5). Bacterial plasmids, both low and high copy, also have specific cellular addresses and segregate in a fashion that is unique for a given plasmid (6-8).Little is known, however, about RNA dynamics in bacteria. With the advent of new methods to label RNA in live cells, the transcription kinetics, localization, and movement of RNA in the bacterium Escherichia coli has begun to be discerned only recently (9-13).To understand RNA dynamics in live cells better, it would be useful to develop RNA labeling methods that would allow direct visualization and real-time quantitation of RNAs with low background levels. We recently reported a system based on protein complementation that uses binding of a split and inactive protein complex to a short interacting sequence on a target RNA. The marker protein re-associates and becomes fluorescent only upon binding to RNA (13), which makes this approach more desirable than alternative techniques relying on expression of full-size fluorescent proteins. Briefly, the method consists of fusing the Nterminal fragment of EGFP to the N-terminal domain of an RNA-binding protein, the eukaryotic initiation factor 4A (eIF4A), via a polypeptide linker. Similarly, the C-terminal fragment of EGFP is fused to the C-terminal domain of eIF4A. The target RNA is tagged at the 3Ј end with an aptamer sequence known to bind eIF4A with high affinity (14) (Fig. 1A). Expression of the labeling components in E. coli cells generates a fluorescent signal only in the presence of the target RNA, caused by the reassociation of the two EGFP fragments and forma...