GLUTAMIC ACID-371 OF THE BARNASE HOMOLOGY DOMAIN IN RNA-POLYMERASE-IIIS NOT REQUIRED FOR SII-ACTIVATED RNA CLEAVAGE

RNA polymerase II contains a ribonuclease activity which is stimulated by the transcription elongation factor SII. This nuclease shortens th e nascent RNA and facilitates relief of transcriptional arrest by allo wing: the enzyme to make multiple attempts to read through an obstacle to transcription. The catalytic center of this ribonuclease is unknow n, although a region of the enzyme's second largest subunit shares loc al sequence similarly with barnase and other bacterial ribonucleases. To test the role of the barnase homology region in SII-activated cleav age, we engineered a single amino acid change in the Saccharomyces cer evisiae enzyme at a position homologous to a catalytic residue of barn ase (Glu-371) and has been suggested as a participant in active site c hemistry of RNA polymerase II. We purified RNA polymerase II from muta nt yeast and assayed its ability to cleave and re-extend the nascent R NA following SII treatment. We find no defects in this function of the mutant enzyme, suggesting that the barnase homology region does not r epresent the active site of the SII-activated nuclease. These mutant y east cells were also resistant to mycophenolic acid, which slows the g rowth of some yeast mutants bearing elongation defective RNA polymeras e II or mutant elongation factor SII.