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23S ribosomal RNA is the large ribosomal RNA component of the 50S large ribosomal subunit in bacteria and archaea. It forms much of the subunit's structural framework and the RNA-based environment of the peptidyl transferase center. The functional RNA is distinct from the DNA sequence from which it is transcribed: a genomic sequence record and a mature ribosomal component describe different physical molecules.^[ref]

Structural experiments with an archaeal large ribosomal subunit placed two substrate analogues against conserved regions of 23S ribosomal RNA. The absence of nearby protein side chains at the bond-forming position supported the conclusion that the ribosome is a ribozyme.^[ref]

The 23S label refers to a sedimentation coefficient, not 23 nucleotides. It must not be added as though it were a molecular mass: the 50S large ribosomal subunit contains 23S ribosomal RNA, 5S ribosomal RNA and ribosomal proteins, and its sedimentation depends on the assembled object's shape and resistance to motion.^[ref] Thus a measurement of an isolated RNA component and a measurement of the complete subunit need not carry the same S value.

5S ribosomal RNA is a small, roughly 120-nucleotide ribosomal RNA of the large subunit of cytoplasmic ribosomes. Its folded RNA contributes to the central protuberance, together with ribosomal proteins and parts of the larger ribosomal RNA. In bacterial ribosomes, it associates with the 50S subunit. The name refers to a sedimentation coefficient, not a count of five bases. Its conserved fold contains five helices and five loops.^[ref]

The role of a component can be tested by changing its physical independence. In a 2020 experiment, researchers joined a rearranged 5S sequence to the much larger 23S ribosomal RNA and removed the ordinary free 5S copies from Escherichia coli. Some engineered cells survived and their properly assembled ribosomes remained active in translation. However, defective large subunits accumulated. Cryo-electron microscopy showed malformed regions of the catalytic center in these misassembled particles.^[ref] Survival therefore distinguishes the need for the 5S sequence from the need to keep it as a separate RNA; it does not establish that assembly is unaffected.

In eukaryotes, 5S ribosomal RNA is transcribed by RNA polymerase III, unlike the major precursor transcribed by RNA polymerase I. The 1974 polymerase study identified the polymerase-III association through distinct inhibition responses of RNA synthesis in isolated nuclei and separated enzyme preparations.^[ref]

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.

Shijie Huang is the first author of a 2020 study testing why 5S ribosomal RNA is normally an independent molecule within the ribosome. The researchers engineered a covalent fusion of rearranged 5S RNA with 23S RNA and produced bacterial cells without free 5S RNA.^[ref]

The engineered cells remained viable under the tested standard conditions, and their fully assembled ribosomes could perform translation. However, they accumulated malformed large subunits that failed to form stable complete ribosomes. Cryo-electron microscopy revealed defects near the peptidyl transferase center in these misassembled particles.^[ref]

This separates two questions: whether an altered ribosome can function once assembled, and whether it assembles efficiently. The results support an assembly-related reason for preserving an independent 5S RNA. Viability in one laboratory condition does not establish that the fusion would be equally successful across environments or evolutionary timescales.