Diagram the elution pattern of the eukaryotic nuclear RNA...

Diagram the elution pattern of the eukaryotic nuclear RNA polymerases from DEAE-Sephadex chromatography. Show what you would expect if you assayed the same fractions in the presence of $1 \mu \mathrm{g} / \mathrm{mL}$ of $\alpha$-amanitin.

Key Concepts

Elution Pattern Analysis

The elution pattern in a chromatography experiment reflects the order in which molecules disassociate from the matrix based on their charge interactions. When combining the chromatography separation with the presence of an inhibitor like alpha-amanitin, the observed enzyme activities and peaks in the collected fractions are altered; the inhibited polymerase(s) will show reduced or absent activity, while resistant polymerases will continue to display their characteristic elution profile. This approach helps in correlating biochemical activity with the identity of the enzyme present in each fraction.

Alpha-Amanitin Inhibition

Alpha-amanitin is a potent cyclic peptide toxin known for its high affinity for certain eukaryotic RNA polymerases. It specifically inhibits RNA polymerase II at low concentrations and also affects RNA polymerase III at higher concentrations, while RNA polymerase I shows relative resistance. This selective inhibition is a useful biochemical tool for distinguishing between the activities of these polymerases in an assay.

DEAE-Sephadex Chromatography

This is an ion-exchange chromatography technique that utilizes the diethylaminoethyl (DEAE) functional groups attached to Sephadex beads. Molecules are separated based on their charge properties; those that are more negatively charged bind more tightly to the positively charged DEAE groups and elute later when a salt gradient is applied. The technique is widely used for protein purification and fractionation.

Eukaryotic Nuclear RNA Polymerases

Eukaryotic cells possess multiple RNA polymerases (primarily RNA polymerases I, II, and III) that are responsible for transcribing different classes of genes. Each polymerase has unique properties, functions, and binding characteristics, which can be exploited in biochemical assays and chromatography to separate them based on their differing interactions with ion-exchange media or inhibitors.

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