Abstract Transcription is the critical first step in expressing a gene, during which an RNA polymerase (RNAP) synthesizes an RNA copy of one strand of the DNA that encodes a gene. Here we describe a laboratory experiment that uses a single assay to probe two important steps in transcription: (1) RNAP binding to DNA, and (2) the transcriptional activity of the polymerase. Students probe both these steps in a single experiment using a fluorescence‐based electrophoretic mobility shift assay (EMSA) and commercially availableEscherichia coliRNAP. As an inquiry‐driven component, students add the transcriptional inhibitor rifampicin to reactions and draw conclusions about its mechanism of inhibition by determining whether it blocks polymerase binding to DNA or transcriptional activity. Depending on the curriculum and learning goals of individual courses, this experimental module could be easily expanded to include additional experimentation that mimics a research environment more closely. After completing the experiment students understand basic principles of transcription, mechanisms of inhibition, and the use of EMSAs to probe protein/DNA interactions.
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Density- and elongation speed-dependent error correction in RNA polymerization
Abstract Backtracking of RNA polymerase (RNAP) is an important pausing mechanism during DNA transcription that is part of the error correction process that enhances transcription fidelity. We model the backtracking mechanism of RNAP, which usually happens when the polymerase tries to incorporate a noncognate or ‘mismatched’ nucleotide triphosphate. Previous models have made simplifying assumptions such as neglecting the trailing polymerase behind the backtracking polymerase or assuming that the trailing polymerase is stationary. We derive exact analytic solutions of a stochastic model that includes locally interacting RNAPs by explicitly showing how a trailing RNAP influences the probability that an error is corrected or incorporated by the leading backtracking RNAP. We also provide two related methods for computing the mean times for error correction and incorporation given an initial local RNAP configuration. Using these results, we propose an effective interacting-RNAP lattice that can be readily simulated.
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- Award ID(s):
- 1814364
- PAR ID:
- 10352198
- Date Published:
- Journal Name:
- Physical Biology
- Volume:
- 19
- Issue:
- 2
- ISSN:
- 1478-3967
- Page Range / eLocation ID:
- 026001
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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