RNA polymerase II subunit Rpb9 regulates transcription elongation in vivo

Area of Science:

• Molecular Biology
• Yeast Genetics
• Transcription Regulation

Background:

• RNA polymerase II (Pol II) is the central enzyme for transcribing protein-coding genes.
• The Rpb9 subunit of Pol II is known to influence transcription initiation and elongation.
• Transcription elongation factor TFIIS plays a critical role in processing stalled transcription complexes.

Purpose of the Study:

• To investigate the specific cellular role of the yeast RPB9 subunit in transcription.
• To determine whether RPB9's primary function relates to transcription initiation or elongation.
• To analyze the functional domains of RPB9 using site-directed and deletion mutants.

Main Methods:

• Yeast genetics: construction and analysis of RPB9 deletion and site-directed mutants.
• Phenotypic analysis: assessing growth defects, 6-azauracil sensitivity, and transcription initiation site selection.
• Complementation studies: evaluating mutant RPB9 function in vivo.
• Genome-wide transcription profiling: analyzing gene expression changes in Deltarpb9 cells.

Main Results:

• RPB9 deletion causes sensitivity to 6-azauracil, a phenotype linked to transcription elongation defects.
• The N-terminal zinc ribbon of RPB9 is important for correct transcription initiation site selection.
• Mutants affecting elongation primarily complemented elongation defects, suggesting elongation is a key role.
• Genome-wide profiling revealed minor changes in metabolic genes in Deltarpb9 cells.

Conclusions:

• RPB9 plays a significant role in transcription elongation, evidenced by synthetic lethality with TFIIS mutations and 6-azauracil sensitivity.
• While RPB9 influences initiation site selection, its primary cellular function appears to be in transcription elongation.
• Specific domains within RPB9 contribute differentially to initiation and elongation processes.