RNA Polymerase II Trigger Loop Mobility: INDIRECT EFFECTS OF Rpb9

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• RNA polymerase II (pol II) is crucial for gene transcription.
• The Rpb9 subunit of pol II affects enzymatic properties and transcription fidelity.
• Rpb9's role in regulating the trigger loop (TL) mobility has been proposed but mechanistically undefined.

Purpose of the Study:

• To elucidate the biochemical mechanism by which Rpb9 influences RNA polymerase II (pol II) function.
• To investigate the interaction between Rpb9 and the Rpb1 subunit, specifically concerning the trigger loop (TL).
• To identify genetic elements that modify the effects of Rpb9 on pol II activity.

Main Methods:

• Utilized the mushroom toxin α-amanitin to probe the role of TL mobility.
• Generated and analyzed missense alleles of RPB9 in yeast.
• Assessed in vitro transcription elongation rates and nucleotide misincorporation rates.

Main Results:

• α-amanitin suppressed Rpb9's effect on nucleotide misincorporation, supporting Rpb9's role in this process.
• Identified yeast RPB9 alleles that suppress growth defects caused by an RPB1 mutation (rpb1-G730D).
• Disrupting proposed Rpb9-Rpb1 interactions led to phenotypes similar to Rpb9 deletion, including increased elongation rate.

Conclusions:

• Rpb9 indirectly modulates TL mobility by anchoring Rpb1's α-helix 21 (α21).
• Rpb9 facilitates intra-pol II interactions that regulate TL function.
• These interactions are critical for controlling pol II enzymatic properties and transcription fidelity.