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Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach
12:12

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

Published on: March 12, 2017

Novel role for mediator complex subunit Srb5/Med18 in termination of transcription

Banupriya Mukundan1, Athar Ansari

  • 1Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202, USA.

The Journal of Biological Chemistry
|September 17, 2011
PubMed

Insights

The Mediator complex

Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Yeast Genetics

Background:

  • The Mediator complex is a crucial transcriptional co-activator.
  • Its known roles include transcription initiation and post-initiation steps.
  • A role in transcription termination remained largely unexplored.

Purpose of the Study:

  • To investigate a potential novel role of the Mediator complex in transcription termination.
  • To identify specific Mediator subunits involved in this process.
  • To elucidate the molecular mechanisms underlying Mediator-dependent termination.

Main Methods:

  • Chromatin cross-linking assays to detect Mediator subunit interactions.
  • Analysis of transcription factor recruitment to gene promoters.
  • Assessment of cleavage-polyadenylation factor association at gene 3' ends.
  • RNA polymerase II localization studies.
  • Transcription run-on assays to detect readthrough transcription.

Main Results:

  • Mediator subunit Srb5/Med18 was found to cross-link to the 5' and 3' ends of specific genes (INO1, CHA1).
  • In the absence of Srb5/Med18, recruitment of TATA-binding protein (TBP) and transcription factor IIB (TFIIB) was unaffected.
  • However, association of cleavage-polyadenylation factors (Rna15, Pta1) with gene 3' ends was compromised.
  • RNA polymerase II accumulated near and beyond the 3' ends of genes, indicating a transcription readthrough phenotype.
  • Transcription run-on assays confirmed this readthrough in srb5(-) cells.

Conclusions:

  • The Mediator subunit Srb5/Med18 plays a critical role in the termination of transcription for a subset of genes in budding yeast.
  • Srb5/Med18 is required for the proper recruitment of termination factors, not initiation factors.
  • Disruption of Srb5/Med18 function leads to transcription readthrough, highlighting its importance in regulating gene expression termination.

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