The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5'-untranslated part of mRNA.
Bottom Line: All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site.For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability.The two UTR sequences also stimulated expression from a constitutive σ(70)-dependent promoter (P1/P(anti-tet)), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription.
Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.Show MeSH
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Mentions: As understanding of the parameters controlling the levels of gene expression is obviously of very general importance in biology and biotechnology, we wanted to confirm the conclusion above by an alternative and independent type of experiment. To achieve this we studied the kinetics of transcript induction from time zero, immediately before the inducer is added, and up to the point where transcript accumulation had ceased. If the rate of bla transcript formation is increased in the cells containing the mutated UTR sequence, one would expect the kinetics of accumulation to be faster in the mutant strain than in the wild type from just after induction and continuously up to an equilibrium state where no further accumulation per unit of cell mass would take place. In contrast, if the observed transcript amounts were the result of enhanced transcript stability, one would expect the rates of accumulation to be similar at early stages after induction, while accumulation would continue for much longer time for the strain containing the mutated UTR sequence than for the strain with the wild‐type sequence. We found that qRT‐PCR was a very good and reproducible method to quantify the kinetics of transcript accumulation. By comparing the accumulation of the bla transcript from cells with plasmid pIB11 (wild‐type UTR) and the corresponding plasmid containing the LV‐2 UTR, a very clear conclusion could be drawn (Fig. 5). The transcript accumulation rate is significantly higher for the strain with the LV‐2 UTR compared with the strain with the corresponding wild‐type sequence, with an approximately 14‐fold higher final accumulation of transcript, consistent with previous estimates (Fig. 2B). However, the time required to reach the point where accumulation levels off is nearly identical (approximately 20 min). Transcript levels are a balance between transcription rate and decay of individual transcripts, the latter determining the time scale for equilibration after promoter induction. Thus, the similar time scales for transcript level equilibration strongly suggests that transcript stability is similar in these two constructs. This was further analysed by fitting a simple model for transcript accumulation (see Supporting Information), where we found no evidence for a difference in transcript stability (P > 5%), average lifetime 10 min, but found a highly significant increase (P < 1%), by at least 18‐fold, in the transcription rate in the LV‐2 UTR strain. Thus, this kinetics analysis provides strong evidence that the rate of transcript formation has increased, and the results of this experiment are consistent with the tRNAArg5 fusion experiment.
Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.