Characterization of two types of termination signal for bacteriophage T7 RNA polymerase.
The late bacteriophage T7 terminator (T7-T phi) 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 the stem-loop and U-tract are not sufficient for termination, and that sequences upstream from the terminator have marked effects on the position and efficiency of termination. 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 site of 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 required for 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 of T7 RNA polymerase resulting from a single proteolytic cleavage 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-T phi.
Macdonald
L E
LE
Department of Microbiology and Immunology, Suny-Health Science Center, NY 11203-2098.
Durbin
R K
RK
Dunn
J J
JJ
McAllister
W T
WT
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010281:Parathyroid Hormone / Q000235:genetics; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics; D017384:Sequence Deletion; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic / Q000502:physiology*; D014764:Viral Proteins
D010281:Parathyroid Hormone; D011498:Protein Precursors; D014764:Viral Proteins; C019056:preproparathormone; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D015719:Single-Strand Specific DNA and RNA Endonucleases