The new super-resolution fluorescence methods enable location and tracking of specific biomolecules in live cells, providing spatiotemporal information at an unprecedented level of detail. We have located and tracked single ribosomes (chromosomally expressed S2-mEos2), RNA polymerase copies (chromosomally expressed b’-mEos2), HU-mEos2 (from a plasmid), and DNA loci (ParB-XFP labeling) in live E. coli with spatial accuracy of s ~ 30 nm. Ribosome-RNAP segregation is strong, arguing against co-transcriptional translation as the primary means of protein synthesis. Diffusion of both ribosomes and RNAP is heterogeneous, enabling us to distinguish translating 70S ribosomes from 30S copies searching for translation initiation sites and transcribing RNAP copies from those searching for transcription initiation sites. In fast growth conditions, the transverse distribution of transcribing RNAP copies is substantially wider than that of HU-mEos2, suggesting that rrn operons preferentially locate near the nucleoid periphery. Transcription of genes coding for proteins is evidently more uniformly distributed throughout the nucleoid. The data also provide circumstantial evidence in favor of the transertion hypothesis.