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Analysis of RNA Polymerase II Mutants using Roche’s xCELLigence System

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The C-terminal domain (CTD) of RNA Polymerase II consists of multiple repeats of an evolutionary conserved motif of seven amino acids, forming a repetitive structure of 52 repeats in humans and 26 repeats in yeast. Twenty years after the first description of the RNA polymerase II C-terminal domain, its function is still unclear.

In a recent research study, M. Heidemann used Roche’s xCELLigence RTCA SP Instrument for in vitro testing of cells to test entirely synthetic CTDs.

According to his findings, the new xCELLigence System technology allows for the first time the monitoring of cell proliferation and cell death throughout the entire experiment, constantly recording cellular effects during cell plating, transfection, and compound treatment. This non-invasive and continuous monitoring of cells provides a far more detailed picture into the whole experimental process, revealing both specific and off-target effects that may have been missed using conventional endpoint assays.

The precise regulation of gene expression is a very important feature of how organisms respond to environmental changes and regulate cell proliferation, development, and even programmed cell death. Gene expression starts with the transcription of genomic DNA into messenger RNA, the template for protein synthesis during ribosomal translation. RNA polymerase Pol II is one out of three different RNA polymerases used by eukaryotic cells to produce the main categories of cellular RNA.

In contrast to Pol I and Pol III, Pol II transcripts are translated into proteins. Pol II is also modified by enzymes influencing different stages of transcription, processing of premature mRNAs in a promoter-dependent manner. These modifications occur in a domain at the carboxy-terminal part of the largest subunit of Pol II, which has a very unique structure.