The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient in Escherichia coli but its transcripts are poorly expressed.
In gene expression studies, promoters are often fused to a protein-encoding reporter gene, the expression of which is then taken as an indirect measure of their strength. Here, we advocate the use of a tRNA reporter for the direct quantification of promoter strength. Using this method, we have studied the bacteriophage T7 gene 10 promoter in an E. coli strain that produces saturating amounts of T7 RNA polymerase. At 37 degrees C in aminoacid-glycerol medium, we show that this promoter ranks amongst the strongest known, directing ca 1.1 transcription events per second, 2.2-fold more than the promoters for rRNA operons, or 15-fold more than the induced lac promoter. Surprisingly, compared to the lac promoter, the T7 promoter is far less efficient in driving the expression of protein-encoding genes such as cat, neo or lacZ. Therefore, the polypeptide yield per transcript is lower when the T7 RNA polymerase is used instead of the E. coli RNA polymerase. The former enzyme travels faster than the translating ribosomes, and we suggest that this desynchronization lowers the polypeptide yield per transcript.
Lopez
P J
PJ
Laboratoire de Génétique Moléculaire, CNRS D1302, Ecole Normale Supérieure, Paris, France.
Iost
I
I
Dreyfus
M
M
D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D017930:Genes, Reporter; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D012343:RNA, Transfer / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases