The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient inEscherichia colibut its transcripts are poorly expressed

Abstract

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 370C 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. INTRODUCTIONBacteriophages T3, T7 and SP6 encode closely related RNA polymerases which transcribe the viral genome from highly specific promoters, during the late phase of infection (1). The prototype of the group, the T7 RNA polymerase, can be stably expressed in E. coli, and genes that are fused to a T7 late promoter can be expressed to high levels when introduced in such hosts (2, 3). In spite of this practical bearing, relatively little is known about the in vivo behaviour of these RNA polymerases. Indirect evidence suggests that the T7 RNA polymerase interacts very efficiently with its promoter in E. coli (4), whereas in vitro the interaction appears relatively weak (1,5). The enzyme has a very high turnover for the polymerisation of ribonucleotides, both in vitro (6) and in vivo (7). As a result, in E. coli it runs far ahead of the ribosomes which translate its nascent transcript, a situation

AU-Rich Sequences within 5′ Untranslated Leaders Enhance Translation and Stabilize mRNA in Escherichia coli

“…To quantify the steady-state level of lacZ mRNA, aliquots of the cultures used for ␤-galactosidase measurements were collected, and total RNA was isolated essentially as described previously (12). RNA samples (10 g) were separated on 1.6% agarose-formaldehyde gels and vacuum blotted onto nylon membranes (21). To visualize the lacZ transcripts, the membrane was probed with the 1.8-kb HincII fragment of lacZ uniformly labeled with 32 P (internal probe).…”

“…Hybridization signals specific for 23S rRNA served as an internal standard to correct lane-to-lane variations in RNA loading. All procedures were carried out as described by Lopez et al (21). Hybridization signals were quantified with a Fuji FLA3000 Imager.…”

The Histone-Like Protein HU Does Not Obstruct Movement of T7 RNA Polymerase in Escherichia coli Cells but Stimulates Its Activity

“…The extraction of RNA, its separation on agarose or acrylamide gels, and its blotting onto a nylon membrane were as described elsewhere, as is the probing of tRNA Arg5 and 5S rRNA with complementary 32 P-labeled oligonucleotides (33,35). The lacZ mRNA was probed with a uniformly 32 Plabeled 1.8-kb HincII fragment internal to the lacZ gene (33).…”

“…To assess the effect of HU on the activity of T7 RNAP in E. coli, we have exploited the techniques available for measuring accurately the level of transcription through the E. coli chromosome (35) and for manipulating the concentration of HU in vivo (54). We report here that the presence of putative HU obstructers has no detectable effect on transcription elongation.…”

“…1. Strain ENS305 (this work) differs from ENS0305 (33) by the presence of a chromosome-borne T7 RNAP gene; it was constructed similarly, except that MO20 (35) was the recipient strain in the final transduction.…”