Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells. Importance of the 5' untranslated leader.
We have analyzed the structure and stability of RNA synthesized by bacteriophage T7 RNA polymerase in mammalian cells. The T7 polymerase, expressed by a recombinant vaccinia virus, transcribed the Escherichia coli lacZ gene flanked by T7 promoter and terminator signals. The lacZ gene cassette was introduced into infected cells within either a transfected plasmid or a second recombinant vaccinia virus. The T7-lacZ transcripts, which had a half-life of approximately 75 minutes, represented approximately 30% of total cytoplasmic RNA after a 24 hour period. The latter estimation indicated a disparity between the levels of lacZ RNA and beta-galactosidase synthesis. Analysis of the T7 transcripts indicated that they were initiated correctly but that only 5 to 10% contained terminal cap structures, providing an explanation for the low translatability of the RNA. Since the 5' end of the T7 transcripts can form a stem-loop structure that might interfere with capping by vaccinia virus RNA guanylyltransferase, as well as ribosome binding and scanning, a similar vector lacking such sequences was constructed. In vitro experiments demonstrated that T7 RNA polymerase transcribed both templates with similar efficiency and that the RNA lacking the potential to form the stem-loop was capped more rapidly by the purified vaccinia virus enzyme. Nevertheless, when the stem-loop was removed, beta-galactosidase was not expressed in infected cells; moreover, no T7 transcripts could be detected, suggesting that the RNA was not made or more likely was degraded during or shortly after synthesis. There is previous evidence that vaccinia virus RNA guanylyltransferase is associated with the viral transcription complex, thereby allowing RNA synthesis and capping to occur concurrently. We suggest that a lack of coupling between the vaccinia viral RNA guanylyltransferase and bacteriophage T7 RNA polymerase delays capping of T7 transcripts and that, under these conditions, the 5'-terminal double-stranded stem is required to stabilize the nascent RNA against degradation. Although deletion of the 3' palindromic sequence specifying T7 transcriptional termination from the expression cassette resulted in RNA of more heterogeneous lengths, neither the apparent turnover rate nor translation of the RNAs was diminished appreciably.
Fuerst
T R
TR
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
Moss
B
B
D000818:Animals; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005814:Genes, Viral; D006367:HeLa Cells; D007763:Lac Operon; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D013604:T-Phages / Q000201:enzymology*; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000378:metabolism*; D001616:beta-Galactosidase / Q000032:analysis
D012333:RNA, Messenger; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase