Specific transcriptional pausing activates polyadenylation in a coupled in vitro system

Area of Science:

• Molecular Biology
• Gene Expression
• RNA Processing

Background:

• RNA polymerase II (Pol II) termination is crucial for gene expression.
• Termination is linked to the polyadenylation of nascent RNA.
• The role of specific DNA sequences in coupling transcription and polyadenylation is not fully understood.

Purpose of the Study:

• To investigate the mechanism of Pol II termination.
• To determine the role of G-rich sequences and transcription factor MAZ in RNA processing.
• To explore the relationship between Pol II pausing and polyadenylation.

Main Methods:

• Development of a coupled in vitro transcription-polyadenylation system.
• Analysis of G-rich sequences' effect on Pol II pausing and polyadenylation.
• Comparison with uncoupled cleavage assays and pausing induced by other proteins.

Main Results:

• G-rich sequences, identified as MAZ binding sites, pause Pol II and activate upstream polyadenylation.
• These sequences specifically enhance polyadenylation, not affecting uncoupled cleavage.
• Pol II pausing induced by other means did not activate polyadenylation, highlighting the specific role of G-rich sequences.
• G-rich sequences promote intrinsic Pol II pausing.

Conclusions:

• G-rich sequences play a specific role in activating polyadenylation.
• These sequences modulate cotranscriptional RNA processing events by inducing pausing.
• The findings reveal a mechanism linking transcription pausing to RNA polyadenylation and termination.