The T7 concatemer junction sequence interferes with expression from a downstream T7 promoter in vivo.
A recently described new signal for transcription termination in vitro by T7 RNA polymerase has now been tested in vivo. This signal, identified during transcription of the cloned human preproparathyroid hormone (PTH) gene, is also found in the phage T7 genome, at the concatemer junction (CJ). We introduced the 17-bp concatemer junction sequence at the ends of a test gene and control gene (both derived from T7 gene 9) in a T7 vector previously used to study effects of rare codons on expression. The CJ elements replaced the original vector's RNase III processing sites, and a new T7 promoter was also introduced to drive the downstream (control) gene. We assayed for test and control gene mRNA and protein by direct labeling with [32P]phosphate and [35S]methionine. The altered vector with CJ sequences (pCT1.1) expressed the upstream test gene, but showed poor expression of the downstream control gene. No discrete T7 mRNA bands could be discerned by direct labeling with 32P. A precursor vector with only the control gene in single copy expressed the protein much better, suggesting that the inhibition of control gene expression in pCT1.1 was a result of the upstream CJ element at the 3' end of the test gene. RT-PCR experiments were consistent with readthrough and possibly pausing at CJ. An RNA folding program predicts a highly stable secondary structure between the upstream CJ element and the control gene's translation start signals. These data support an interpretation that the CJ element is ineffective as a T7 transcription terminator in vivo in this vector, and that structure of the readthrough transcript blocks ribosome access to the downstream translation start. The readthrough transcripts are also likely to be less stable than properly terminated or processed T7 mRNA, because levels of test protein expression in pCT1.1 were reduced compared to original vector, and basal expression was negligible, while the original codon test vector shows substantial basal expression.
Harvey
B
B
Graduate School of Biomedical Sciences, University of Medicine & Dentistry of New Jersey, Newark 07103, USA.
Korus
M
M
Goldman
E
E
D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D003062:Codon; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013728:Terminator Regions, Genetic; D014764:Viral Proteins
D003062:Codon; D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases