Inhibition of RNA polymerase III transcription by a ribosome-associated kinase activity

Area of Science:

• Molecular Biology
• Gene Regulation
• Xenopus laevis research

Background:

• Ribosomes from Xenopus laevis somatic tissue inhibit RNA polymerase III transcription.
• This mirrors previous findings with ribosome-enriched fractions from egg extracts affecting RNA polymerase III activity and transcription complexes.
• Both somatic and oocyte 5S rRNA genes, as well as tRNA genes, are repressed.

Purpose of the Study:

• To identify the specific component of ribosomes responsible for inhibiting RNA polymerase III transcription.
• To determine the mechanism by which ribosomes repress class III gene transcription.

Main Methods:

• DNase I protection assays to analyze transcription complex formation.
• High salt extraction to isolate factors associated with ribosomes.
• Enzyme treatments (proteinase K, micrococcal nuclease) to characterize the inhibitory factor.
• Western blot analysis to detect specific proteins, including p34(cdc2).
• Use of a protein kinase inhibitor (6-dimethylaminopurine).

Main Results:

• The TFIIIA and TFIIIC complex formation on 5S rRNA genes is stable and not inhibited by ribosomes.
• High salt treatment of ribosomes abolishes their inhibitory activity, indicating an associated factor.
• The inhibitory activity is sensitive to proteinase K but not micrococcal nuclease.
• A protein kinase inhibitor prevents ribosome-mediated repression.
• p34(cdc2), a known repressor of RNA polymerase III transcription, is present on ribosomes and can be released by high salt.

Conclusions:

• The inhibition of RNA polymerase III transcription by Xenopus laevis ribosomes is mediated by an associated kinase activity.
• This kinase activity, likely p34(cdc2), is the actual repressor, not the ribosome particle itself.
• The findings elucidate a novel regulatory mechanism for class III gene transcription involving ribosomal-associated factors.