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RNA polymerase III is a eukaryotic RNA polymerase that transcribes several small functional RNAs, including transfer RNA and 5S ribosomal RNA. It is distinct from RNA polymerase I, which makes the large precursor of most other ribosomal RNAs, and from RNA polymerase II, which transcribes protein-coding genes and many noncoding RNAs.

In 1974, Roberto Weinmann and Robert G. Roeder compared inhibition of RNA synthesis in isolated nuclei with the characteristic inhibitor sensitivity of separated RNA polymerases. The behaviour of transfer RNA and 5S ribosomal RNA synthesis matched RNA polymerase III, assigning particular RNA products to an enzymatic activity rather than grouping all nuclear transcription together.^[ref] This division of labour explains how production of translation machinery can be regulated separately from production of messenger RNA.

Robert G. Roeder investigates transcription and its regulation. His 1974 study with Roberto Weinmann helped identify which RNA polymerase produces transfer RNA and 5S ribosomal RNA. The researchers compared sensitivity to alpha-amanitin in transcription by isolated cell nuclei with the sensitivities of separated polymerase activities.^[ref]

Matching these responses supported the role of RNA polymerase III. The experiment connected production of particular RNA products to a biochemical activity, instead of assuming that every gene in a cell nucleus is transcribed by the same enzyme.

Roberto Weinmann coauthored a 1974 study assigning transcription of transfer RNA and 5S ribosomal RNA genes to RNA polymerase III. With Robert G. Roeder, he compared the effects of alpha-amanitin on RNA synthesis in isolated cell nuclei and on chromatographically separated polymerase activities.^[ref]

The logic was comparative: an inhibitor-response pattern for a defined RNA product could be matched to a separated enzyme activity. This linked a biochemical distinction between RNA polymerases to the kinds of genes they transcribe, rather than inferring function from enzyme abundance alone.