Question

a. How could you experimentally determine whether a purified preparation of an RNA polymerase is from a prokaryotic or eukaryotic source? b. A purified preparation of RNA polymerase is sensitive to inhibition by \(\alpha\) -amanitin at a concentration of \(10^{-8} \mathrm{M}\). The synthesis of what type or types of RNA is inhibited?

Short answer

Answer: At a concentration of \(10^{-8} \mathrm{M}\), α-amanitin would inhibit the synthesis of messenger RNA (mRNA) and small nuclear RNA (snRNA). To determine the source of a purified RNA polymerase sample, an experimental approach could involve testing the purified preparation against specific inhibitors, such as rifampicin and streptolydigin for prokaryotic RNA polymerases, or α-amanitin for eukaryotic RNA polymerases, and assaying for the remaining activity.

Step-by-step solution

Part a: Distinguishing between prokaryotic and eukaryotic sources of RNA polymerase

To determine the origin of the purified RNA polymerase, we can consider analyzing the properties of both prokaryotic and eukaryotic RNA polymerases or their sensitivity towards specific inhibitors. Prokaryotic cells, such as bacteria, contain a single type of RNA polymerase, which synthesizes all types of RNA molecules. Eukaryotic cells, on the other hand, contain multiple types of RNA polymerases that are responsible for synthesizing different types of RNA molecules. Specifically, in eukaryotic cells: 1. RNA polymerase I is responsible for synthesizing ribosomal RNA (rRNA) 2. RNA polymerase II synthesizes messenger RNA (mRNA) and small nuclear RNA (snRNA) 3. RNA polymerase III synthesizes transfer RNA (tRNA) and other small RNA molecules. An experimental approach to differentiate between prokaryotic and eukaryotic sources of RNA polymerase is to test the purified preparation against specific inhibitors that affect either prokaryotic or eukaryotic RNA polymerases. For example, antibiotics such as rifampicin and streptolydigin selectively inhibit prokaryotic RNA polymerases while having no effect on eukaryotic RNA polymerases. Conversely, α-amanitin, a toxic cyclic peptide, specifically inhibits eukaryotic RNA polymerase II and III (with high and low sensitivity, respectively) but does not affect prokaryotic RNA polymerases. Therefore, by exposing the purified RNA polymerase preparation to these specific inhibitors and assaying for the remaining activity, we can deduce whether the source of RNA polymerase is prokaryotic or eukaryotic.

Part b: Types of RNA synthesis inhibited by α-amanitin

As mentioned earlier, α-amanitin is a potent inhibitor of RNA polymerases in eukaryotic cells. It has different effects on the three types of eukaryotic RNA polymerases depending on the concentration: 1. RNA polymerase I: Insensitive to α-amanitin 2. RNA polymerase II: Highly sensitive to α-amanitin, inhibited by concentrations as low as \(10^{-8} \mathrm{M}\) 3. RNA polymerase III: Inhibited at significantly higher concentrations, typically above \(10^{-6} \mathrm{M}\) Since the given concentration of α-amanitin is \(10^{-8} \mathrm{M}\), only RNA polymerase II will be inhibited in this case. As RNA polymerase II is responsible for synthesizing messenger RNA (mRNA) and small nuclear RNA (snRNA), the synthesis of these two types of RNA will be inhibited.