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Title: Evaluating two steps in transcription using a fluorescence‐based electrophoretic mobility shift assay
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.  more » « less
Award ID(s):
1817442
PAR ID:
10403452
Author(s) / Creator(s):
 ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Biochemistry and Molecular Biology Education
Volume:
51
Issue:
2
ISSN:
1470-8175
Page Range / eLocation ID:
p. 230-235
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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