Abstract
Many nucleic acid polymerases function in clusters known as factories. We investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when active. Using ‘pulldowns’ and fluorescence correlation spectroscopy we find that elongation complexes do not interact in vitro with a Kd<1 µM. Chromosome conformation capture also reveals that genes located 100 kb apart on the E. coli chromosome do not associate more frequently when transcribed by T7 RNAP. We conclude that if clustering does occur in vivo, it must be driven by weak interactions, or mediated by a phage-encoded protein.
Highlights
Mounting evidence suggests that many RNA and DNA polymerases function in clusters rather than in isolation
Mammalian RNA polymerase II (RNAP II), for example, appears to be active in ‘factories’ which typically contain,8 enzymes working on different templates, and DNA polymerases cluster in analogous ‘replication factories’ [1,2,3]
T7 RNAP elongation complex (EC) do not co-associate in vitro To test whether active T7 RNAPs cluster, we examined whether ECs diffusing freely in solution interacted with distinguishable ECs directly attached to beads (Fig. 1A)
Summary
Mounting evidence suggests that many RNA and DNA polymerases function in clusters rather than in isolation. Mammalian RNA polymerase II (RNAP II), for example, appears to be active in ‘factories’ which typically contain ,8 enzymes working on different templates, and DNA polymerases cluster in analogous ‘replication factories’ [1,2,3]. Such ‘factories’ may exist in some [4,5,6] – but perhaps not all [7] – bacteria. Another is that RNAP clustering evolved because freely-mobile enzymes would track along and rotate about their templates, and so entangle their trailing nascent transcripts; RNAPs immobilized in clusters would reel in their templates without rotating, and so extrude unentangled transcripts [11]
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