The Spo0A transcription factor is activated by phosphorylation in starving
RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.
more » « less- NSF-PAR ID:
- 10154276
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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The role of general transcription factor TFIIB in transcription extends well beyond its evolutionarily conserved function in initiation. Chromatin localization studies demonstrating binding of TFIIB to both the 5’ and 3’ ends of genes in a diverse set of eukaryotes strongly suggested a rather unexpected role of the factor in termination. TFIIB indeed plays a role in termination of transcription. TFIIB occupancy of the 3’ end is possibly due to its interaction with the termination factors residing there. Interaction of the promoter-bound TFIIB with factors occupying the 3’ end of a gene may be the basis of transcription-dependent gene looping. The proximity of the terminator-bound factors with the promoter in a gene loop has the potential to terminate promoter-initiated upstream anti-sense transcription thereby conferring promoter directionality. TFIIB, therefore, is emerging as a factor with pleiotropic roles in the transcription cycle. This could be the reason for preferential targeting of TFIIB by viruses. Further studies are needed to understand the critical role of TFIIB in viral pathogenesis in the context of its newly identified roles in termination, gene looping and promoter directionality.more » « less
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Abstract Motivation Transcription by RNA polymerase is a highly dynamic process involving multiple distinct points of regulation. Nascent transcription assays are a relatively new set of high throughput techniques that measure the location of actively engaged RNA polymerase genome wide. Hence, nascent transcription is a rich source of information on the regulation of RNA polymerase activity. To fully dissect this data requires the development of stochastic models that can both deconvolve the stages of polymerase activity and identify significant changes in activity between experiments.
Results We present a generative, probabilistic model of RNA polymerase that fully describes loading, initiation, elongation and termination. We fit this model genome wide and profile the enzymatic activity of RNA polymerase across various loci and following experimental perturbation. We observe striking correlation of predicted loading events and regulatory chromatin marks. We provide principled statistics that compute probabilities reminiscent of traveler’s and divergent ratios. We finish with a systematic comparison of RNA Polymerase activity at promoter versus non-promoter associated loci.
Availability and Implementation Transcription Fit (Tfit) is a freely available, open source software package written in C/C ++ that requires GNU compilers 4.7.3 or greater. Tfit is available from GitHub (https://github.com/azofeifa/Tfit).
Supplementary information Supplementary data are available at Bioinformatics online.
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