Nuclear Structure and Function Research Group

Active RNA polymerases: mobile or immobile molecular machines?

It has been assumed that an RNA polymerase transcribes by diffusing to a promoter, binding, and then tracking down the template as it makes its transcript. We suggested an alternative: a promoter diffuses to a transcription factory where it binds to a transiently-immobilized polymerase, which then reels in its template as it extrudes a transcript (reviewed by Cook, 2010). We now distinguish between the two (Papantonis et al., 2010; pdf; PubMed), and the movie on this page supports this work.

Our strategy involves switching on transcription of one long gene and one short gene using tumor necrosis factor alpha. This cytokine orchestrates the inflammatory response by signaling through nuclear factor kappa B to activate certain genes. In human umbilical vein endothelial cells (HUVECs), a polymerases initiates within ~10 min on SAMD4A – a 221-kbp gene that encodes a regulator of this pathway – and then transcribes at ~3 kbp/min before terminating after ~70 min. A second polymerase initiates as rapidly on TNFAIP2 – an 11-kbp gene lying ~50 Mbp away which encodes another regulator – and then transcribes it repeatedly. We analyze the distances between the two genes (and their transcripts) using 'chromosome conformation capture' (and 'fluorescence in situ hybridization' coupled to 'super-resolution' microscopy).

If the conventional model for transcription applies, TNFAIP2 should not lie close to any part of SAMD4A either before or after adding the cytokine, as the two lie so far apart on the chromosome. Even if polymerases on the two genes happen to lie together (for whatever reason), tracking of one down the long gene should increase inter-polymerase distance. But if both genes are transcribed by polymerases transiently immobilized in one factory, the short gene – which would repeatedly attach to (and detach from) the factory as it initiates (and terminates) – should always lie close to just the part of SAMD4A being transcribed at that particular moment. Thus, as one polymerase reels in SAMD4A, first the promoter, then the middle of the gene, and finally the terminus should be brought into the factory to lie transiently next to TNFAIP2. Results are consistent with the scenario involving active polymerases immobilized in factories.

The movie illustrates the changing contacts between different parts of the two genes.

Movie showing that only transcribed parts of the two genes lie close together Special thanks to Ruth Cammock, Martin Clark, Alaric Kong, Marcel Vieira de Carvalho Nobrega, and Robert Quick who made this animation during an undergraduate project at the University of the West of England. A long region of the genome is diffusing through the nucleoplasm; then, transcription of two genes is switched on synchronously so the long gene is transcribed once and the short gene three times. Another segment of DNA also binds through a transcription factor to the factory. [For the sake of simplicity, (i) this segment remains bound throughout most of the movie (even though we know binding persists for only a few seconds), and (ii) the long and short genes are shown close together (instead of ~50 Mbp apart).] Once a promoter binds, the (now fixed) polymerase reels in its template as it extrudes a transcript. [What happens to the nascent transcript is speculative; here we show it being packaged into a (monomeric) ribonucleoprotein (RNP) and then into a tetrameric large RNP (lnRNP), before it finally diffuses away (Iborra et al., 1998; 2000).] When transcription begins, the short gene lies close to the promoter of the long gene. Later, when the short gene is transcribed for the second time, it lies close to the middle of the long gene (but no longer the promoter). Later still, when the short gene is being transcribed for the third time, it lies close the terminus of the long gene (but no longer the promoter or the middle). Experimental results confirm that the promoter, middle region, and terminus of SAMD4A lie close to TNFAIP2 at the appropriate times after stimulation.

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