A soluble biotinyl-α-amanitin affinity probe for the isolation of RNA polymerase B

Utilizing the 6′-hydroxyindole moiety of α-amanitin for substitution, biotinyl-α-amanitin has been synthesized to use as a soluble affinity probe for the isolation of RNA polymerase B from mammalian cell culture. The synthetic biotinyl-α-amanitin remains a potent inhibitor of RNA polymerase B having a Ki of 4.1 × 10−8 M as compared with a Ki of 5 × 10−9 M for natural α-amanitin. RNA polymerase B complexed with biotinyl-α-amanitin can be isolated on Bio-Gel P300 polyacrylamide gel beads to which avidin has been attached. RNA polymerase B may then be released from the complex by treatment with sodium dodecyl sulfate or by monochromatic irradiation at 314 nm which destroys the amatoxin moiety. We have used this affinity probe to analyze the subunit composition of RNA polymerase B from various mouse myeloma cell lines. We believe that the biotinyl-α-amanitin may be very useful for the isolation of factors which associate with RNA polymerase B; e.g., we have substantiated that actin can be associated with RNA polymerase

Formation of a single phosphodiester bond by RNA polymerase B from calf thymus is not inhibited by alpha-amanitin

The template-directed synthesis of a single phosphodiester bond by highly purified calf thymus RNA polymerase B is not inhibited by high concentrations of alpha-amanitin (10(-6) M). However, a subsequent internucleotide bond is not synthesized in the presence of alpha-amanitin. These results suggest that translocation of the nascent RNA and RNA polymerase B along the DNA template is the enzymatic process inhibited by alpha-amanitin. It is also shown that the formation of a single phosphodiester bond by RNA polymerase B results in a stable ternary transcription complex, i.e., between the enzyme, the DNA, and the nascent RNA. Under reaction conditions which normally favor the elongation of RNA, the transcriptional process is arrested at initiation by alpha-amanitin. Such ternary initiation complexes have been isolated by agarose gel electrophoresis

The Effect of the Chemical Nature of the Side Chains of the Amatoxins in the Inhibition of Eukaryotic RNA Polymerase B

The inhibition constants (Ki) of DNA-dependent RNA polymerase B (or II) from calf thymus were measured for eight synthetically obtained bicyclic amanitin-like thioethers, two R-sulfoxides, and two S-sulfoxides. These Ki values were compared with those of alpha-amanitin, its 6'-O-methylether Ia (an R-sulfoxide), the S-sulfoxide, the sulfone, the S-deoxo derivative (Id) of Ia, and several previously described amatoxins. The necessity of a beta-methyl side chain in position 3 and a hydroxy group in proline-2 was confirmed. Additionally, the presence of an isoleucine side chain in position 6 and the absence of a side chain in position 5 was recognized as important for binding to the enzyme. In the three sulfoxide samples examined, the R-diastereomer was found to be a stronger inhibitor than the S-form. The contribution of every structural element to biological activity has been discussed