Intrinsic transcript cleavage in yeast RNA polymerase II elongation complexes

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Transcriptional roadblocks impede RNA polymerase II progression.
• The transcription factor TFIIS facilitates bypass of these roadblocks via transcript cleavage.
• Yeast RNA polymerase II possesses intrinsic transcript cleavage activity.

Purpose of the Study:

• To investigate the characteristics and functional significance of RNA polymerase II's intrinsic transcript cleavage activity.
• To compare intrinsic cleavage with TFIIS-mediated cleavage.
• To determine if intrinsic cleavage can rescue arrested transcription complexes.

Main Methods:

• Purification of yeast RNA polymerase II.
• In vitro assays of transcript cleavage using arrested and stalled elongation complexes.
• Analysis of small RNA products.
• Testing the effect of pH, divalent cations, and alpha-amanitin on cleavage activity.
• Characterization of a TFIIS-refractory mutant polymerase.

Main Results:

• Yeast RNA polymerase II exhibits intrinsic transcript cleavage activity, stimulated by basic pH and divalent cations.
• This intrinsic cleavage can reactivate some arrested transcription complexes.
• Small RNA products differ between intrinsic and TFIIS-induced cleavage in stalled complexes but are similar in arrested complexes.
• Alpha-amanitin inhibits TFIIS-induced cleavage but not intrinsic cleavage.
• A TFIIS-refractory mutant polymerase shows normal intrinsic cleavage.

Conclusions:

• Intrinsic transcript cleavage by RNA polymerase II is a significant mechanism for overcoming elongation blocks.
• This activity is distinct from TFIIS-mediated cleavage in certain aspects.
• Cleavage is sufficient to reactivate some arrested complexes, highlighting its role in transcription fidelity and progression.