Date of Award

Spring 2001

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Clyde L Denis

Abstract

The CCR4-NOT complex from Saccharomyces cerevisiae, which exists in two forms in vivo: 1.9 and 1.0 x 10 6 MDa in size, is a general transcriptional regulatory complex. It regulates mRNA transcription in initiation, elongation and degradation.

The 1.0 MDa complex was purified to near homogeneity, and mass spectrometric analysis was used to identify all the components of the complex. The 1.0 MDa complex contains CCR4, CAF1, NOT1--5, and two new proteins, CAF40 and CAF130. CAF130 and CAF40 are two unique yeast proteins, with CAF40 displaying extensive homology to proteins from other eukaryotes. Immunoprecipitation and gel filtration experiments confirm that CAF130 and CAF40 are components of both of the 1.9 MDa and 1.0 MDa CCR4-NOT complexes. Biochemical analysis indicated that the CAF40 and CAF130 proteins exist in peripheral position of the complex. CAF130 specifically interacted with both N- and C-terminal domains of the NOT1 protein, suggesting that it lies adjacent to NOT1. Genetic analysis indicated that CAF40 and CAF130 share few phenotypes with the rest of the CCR4-NOT complex genes, consistent with the physical separation of CAF40 and CAF130 from the other proteins in the 1.0 MDa CCR4-NOT complex. CAF40 and CAF130, may, therefore play novel roles in the CCR4-NOT complex.

The CCR4 protein is an evolutionary conserved protein. The strongest conserved region of the CCR4 protein lies in its C-terminal domain, which shows homology with the Mg2+ dependent nuclease family. In this research, we reported that the CCR4 protein indeed displayed 5 ' DNA/RNA phosphatase and 3'--5 ' exonuclease activities. First, we showed that GST-CCR4 (C-terminal domain) protein was able to bind only ssDNA and RNA. Second, we demonstrated that GST-CCR4 (C-terminal domain) protein possessed 5' DNA/RNA phosphatase and 3'--5' exonuclease activities. Third, by using full-length CCR4-FLAG fusion protein, we found that CCR4 displayed a 3'--5' RNA poly (A) exoribonuclease and a poly (dA) ssDNA exonuclease activities, with preference for the RNA substrate. The CCR4 protein with the conserved catalytic residue mutations did not display all these activities. We propose that the regulatory effect of the CCR4-NOT complex on gene expression may be executed through the CCR4 enzymatic function.

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