Characterization of 3' processing ribonucleases CPSF-73 and Rat1/Rai1.

摘要

The Torpedo model of transcription termination requires an endonucleolytic cleavage of pre-mRNA which generates an upstream product that goes on to become the poly(A) tail and a downstream product that is susceptible to attack by 5' to 3' exonucleases. CPSF-73 is the enzyme responsible for endonucleolytic cleavage and in its purified form it requires preincubation with divalent cations for its activity. CPSF-73 possesses two domains a metallo-beta-lactamase and a beta-CASP domain. The position of the beta-CASP domain suggests that it might be the "gate keeper" to obstruct access to the active site. We show that removal of the beta-CASP domain considerably enhanced the nuclease activity of CPSF-73. Our data, taken together with earlier data confirms that CPSF-73 does not function as an exonuclease (Mandel et al. 2006). Furthermore, the yeast 5' to 3' exonuclease Rat1 binds to its activating partner Rai1 on the opposite face of its active site. We show data which strongly suggest that Rai1 stimulates the activity of Rat1 by stabilizing Rat1 binding to RNA. Consistent with earlier data that showed stem loops and oligo (G) tracts can decrease the processivity of Rat1 (Poole and Stevens, 1995; 1997), we also show that Rat1 loses processivity in the presence of hairpin structures and stalls in RNAs. Furthermore, we demonstrate that Rat1/Rai1 possesses activity on 5' triphosphorylated RNA. Our data provides the first structural and biochemical evidence that Rai1 is a RNA triphosphatase.