CHARACTERIZATION OF 2 TYPES OF TERMINATION SIGNAL FOR BACTERIOPHAGE-T7 RNA-POLYMERASE

The late bacteriophage T7 terminator (T7-TΦ)) encodes an RNA sequence that can form a stable stem loop structure followed by a run of six uridylate residues; termination occurs at a 3′ G residue just downstream of the U run. In this work, we have explored the features of this signal that are required for efficient termination by T7 RNA polymerase. Whereas replacement of the template-encoded 3′ G residue with A, C, or U by site-directed mutagenesis had little effect, removal of the U-tract prevented termination. Deletion analysis indicates that thestem-loop and U-tract are not sufficient for termination, and that sequences upstream from the terminator have marked effects on the position and efficiency oftermination. A sequence within the human preproparathyroid hormone (PTH) gene that encodes an interrupted run of six U residues, but lacks an apparent stem-loop structure, also serves as an efficient terminator for T7 RNA polymerase. We have mapped the primary siteof termination in the PTH signal to a G residue that lies downstream of the U-rich run (UUUUCUUG). Deletion analysis indicates that the minimal region requiredfor PTH terminator function extends only 23 bp upstream from the termination site, and subcloning of a 31 bp fragment that includes this region of the PTH signal provides efficient termination. A modified form ofT7 RNA polymerase resulting from a single proteolyticcleavage between residues 178 and 179, or mutant polymerases that are altered in this region of the enzyme, fail to recognize the PTH signal while still terminating at T7-TΦ. © 1994 Academic Press, Inc.