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
Mentions: To distinguish between the hypotheses described above we constructed a transcriptional fusion between the bla gene and a sequence encoding a tRNA (tRNAArg5) such that both genes become expressed as one single transcript from plasmid pRL11 (Fig. 3). The rationale behind this is that the tRNAArg5 part is essentially stable, so that any stimulation of bla transcription would also lead to an increase in the tRNAArg5 formation (Lopez et al., 1994). In contrast, if the increase in bla transcript accumulation is caused by enhanced mRNA stability one would expect essentially no increase in the amounts of tRNAArg5. To test this we used plasmid constructs that were identical, except that the UTR DNA sequence was the wild‐type (pRL11), LV‐1 or LV‐2. It was again, as expected, found that the ampicillin‐resistance levels of the corresponding host cells were much higher for cells containing the mutated UTR DNA sequences compared with those containing the wild‐type sequence. Quantitative real‐time PCR was then used to compare the amounts of bla and tRNAArg5 transcripts (Fig. 4). The amounts of bla transcript were strongly increased (about 11‐fold) in the cells containing the LV‐1 or LV‐2 sequences, relative to those containing the wild‐type sequence, as expected. More interestingly, the amounts of tRNAArg5 had also increased about eightfold. Based on the assumption that the tRNAArg5 part is essentially stable, this appears to confirm that the UTR mutations have resulted in a large increase in the amounts of transcript formed by the RNA polymerase.
Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.