A great challenge for a eukaryotic cell is to produce the appropriate amount of each protein and to quickly adjust that production as the cell’s environment changes.  Much of this balancing act occurs at the levels of mRNA synthesis, translation, and degradation.  Recent work on eukaryotic gene expression has uncovered feedback mechanisms that coordinate these different activities and allow the cell to maintain optimal levels of mRNA. Much of this coordination occurs between the different steps of mRNA synthesis, such that the events of transcription initiation, elongation, and termination are coordinated with the processing steps of capping, splicing, and polyadenylation, and ultimately, with exit from the nucleus.  This “communication network” ensures that each event occurs in a timely manner and also provides additional steps at which the amount and type of mRNA from a particular gene can be regulated. 

Intellectual Merit. In this project, we have probed how polyadenylation factors influence RNA Polymerase II (RNAP II) during the initiation, elongation and termination steps of mRNA transcription. Important outcomes of this project include:

1.  Development of a new, cell-free method to investigate the molecular dynamics that cause termination.  Our findings support the previously proposed model in which termination is provoked by allosteric changes in the transcriptional complex as well as the action of specific enzymes that remove RNAP II from the DNA template.

2.  Identification and characterization of a novel early termination mechanism which regulates the production of full-length mRNAs by generating short, polyadenylated transcripts ending near the gene’s promoter.

3.  Demonstration that termination of transcription when RNAP II is stalled at a DNA lesion requires polyadenylation factors for efficient removal of the blocked polymerase.

4.  Identification of two new domains on the surface of RNAP II that are needed to recruit polyadenylation factors for their function in transcription termination.

5.  Provided evidence that Ysh1, the nuclease that cleaves mRNA precursor at the poly(A) site, is important for transcription elongation.  We also showed that Ipa1, encoded by one of the six essential yeast genes with previously unknown function, serves dual roles of regulating the level of Ysh1 as well as recruiting it to the elongation complex. 

This research resulted in six publications, two manuscripts that have been resubmitted with revisions, and five manuscripts in preparation, as well as presentation of our findings at five different conferences.

Broader Impacts.  In addition to advancing scientific knowledge, this project provided opportunities for training a diverse group of scientists and for encouraging them to integrate their efforts towards common goals. It provided opportunities for faculty at primarily undergraduate institutions (PUIs) to focus on research by spending summers working in our lab and for undergraduates to gain experience in research.  It gave senior lab members experience mentoring younger scientists from widely different backgrounds, and supported the development of new discovery-based lab modules that will be used at local minority-serving colleges.  These initiatives capitalized on connections made possible through our long-standing involvement in minority outreach efforts at the undergraduate, postbaccalaureate, and postdoctoral levels.  This project also facilitated the development of new computational strategies to analyze whole genome data sets and gain insights into the regulation of mRNA polyadenylation and how polyadenylation factors affect transcription initiation.

Last Modified: 07/10/2017
Modified by: Claire L Moore