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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.

Berg L, Lale R, Bakke I, Burroughs N, Valla S - Microb Biotechnol (2009)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.

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Map of plasmid pIB11 and mutations giving rise to stimulated expression of bla. Restriction sites shown are unique. Pm, positively regulated promoter; xylS, gene encoding Pm activator; Apr (bla), ampicillin‐resistance gene encoding β‐lactamase; Kmr, kanamycin‐resistance gene; trfA, gene encoding the essential replication protein; oriV, origin of vegetative replication; oriT, origin of transfer; t, bidirectional transcriptional terminator; rrnBT1T2, bidirectional transcriptional terminator. Details for the DNA sequences corresponding to the Pm 5′‐untranslated transcript region (UTR) are displayed above the plasmid map. SD is the putative Shine–Dalgarno sequence. Nucleotides in lowercase were randomly mutagenized. Deletion mutations are indicated by short horizontal lines. Transcriptional and translational start sites are indicated with an arrow. The following base substitutions were identified in four selected examples from the screening of the LI library (nucleotide numbers are indicated in the 5′ to 3′ direction). Sequence 1: A2C, C3A, A14T. Sequence 2: C3A, A10C, T12C, A14C. Sequence 3: A2T, C3A, G6C, T12C, A14T. Sequence 4: A2T, A8G, C9T, T12C, A14T, T15A. Note that A10C is also found in nine of the 12 LII sequences and T12C is found in four. The LI library was constructed in plasmid pLB1, which differs from pIB11 in the UTR region in that A4 has been changed to T, and T5 to A.
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f1: Map of plasmid pIB11 and mutations giving rise to stimulated expression of bla. Restriction sites shown are unique. Pm, positively regulated promoter; xylS, gene encoding Pm activator; Apr (bla), ampicillin‐resistance gene encoding β‐lactamase; Kmr, kanamycin‐resistance gene; trfA, gene encoding the essential replication protein; oriV, origin of vegetative replication; oriT, origin of transfer; t, bidirectional transcriptional terminator; rrnBT1T2, bidirectional transcriptional terminator. Details for the DNA sequences corresponding to the Pm 5′‐untranslated transcript region (UTR) are displayed above the plasmid map. SD is the putative Shine–Dalgarno sequence. Nucleotides in lowercase were randomly mutagenized. Deletion mutations are indicated by short horizontal lines. Transcriptional and translational start sites are indicated with an arrow. The following base substitutions were identified in four selected examples from the screening of the LI library (nucleotide numbers are indicated in the 5′ to 3′ direction). Sequence 1: A2C, C3A, A14T. Sequence 2: C3A, A10C, T12C, A14C. Sequence 3: A2T, C3A, G6C, T12C, A14T. Sequence 4: A2T, A8G, C9T, T12C, A14T, T15A. Note that A10C is also found in nine of the 12 LII sequences and T12C is found in four. The LI library was constructed in plasmid pLB1, which differs from pIB11 in the UTR region in that A4 has been changed to T, and T5 to A.

Mentions: Initially, a mutated Pm UTR library (LI) was constructed in the corresponding part of the Pm/xylS expression cassette (bla used as reporter gene) by using randomly mutated synthetic oligonucleotides. The mutations were introduced throughout almost the entire UTR sequence and mutants displaying a 10‐fold increase in ampicillin‐resistance levels compared with the wild type, could easily be isolated from this library. DNA sequence analysis showed that they carried three to six mutations in the UTR DNA sequence, none of which affected the putative SD sequence. Four examples of such mutants are indicated in the legend to Fig. 1. Most of the identified sequences had mutations near the transcriptional start site, but as this was not always the case we wanted to specifically investigate how sequences in the internal parts of the UTR affect gene expression. We therefore prepared a new mutated Pm UTR library (LII) in plasmid pIB11 (Fig. 1), based on an oligonucleotide mixture in which only bases more than 7 bp downstream of the transcriptional start site were subjected to random sequence changes. Surprisingly, clones with equally high ampicillin‐resistance levels as in LI could be identified also from this new library. Twelve such mutants were selected for further studies, first by sequencing their UTR DNA sequences (LII‐1 to ‐12), followed by new synthesis of the corresponding oligonucleotides (both strands). These were then exchanged with the corresponding part from the wild‐type pIB11, reconfirmed by DNA sequencing, and phenotype analysed with respect to ampicillin resistance of the hosts, confirming identity with the original mutants isolated. This means that the phenotypes of the mutants are caused solely by the UTR mutations.


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.

Berg L, Lale R, Bakke I, Burroughs N, Valla S - Microb Biotechnol (2009)

Map of plasmid pIB11 and mutations giving rise to stimulated expression of bla. Restriction sites shown are unique. Pm, positively regulated promoter; xylS, gene encoding Pm activator; Apr (bla), ampicillin‐resistance gene encoding β‐lactamase; Kmr, kanamycin‐resistance gene; trfA, gene encoding the essential replication protein; oriV, origin of vegetative replication; oriT, origin of transfer; t, bidirectional transcriptional terminator; rrnBT1T2, bidirectional transcriptional terminator. Details for the DNA sequences corresponding to the Pm 5′‐untranslated transcript region (UTR) are displayed above the plasmid map. SD is the putative Shine–Dalgarno sequence. Nucleotides in lowercase were randomly mutagenized. Deletion mutations are indicated by short horizontal lines. Transcriptional and translational start sites are indicated with an arrow. The following base substitutions were identified in four selected examples from the screening of the LI library (nucleotide numbers are indicated in the 5′ to 3′ direction). Sequence 1: A2C, C3A, A14T. Sequence 2: C3A, A10C, T12C, A14C. Sequence 3: A2T, C3A, G6C, T12C, A14T. Sequence 4: A2T, A8G, C9T, T12C, A14T, T15A. Note that A10C is also found in nine of the 12 LII sequences and T12C is found in four. The LI library was constructed in plasmid pLB1, which differs from pIB11 in the UTR region in that A4 has been changed to T, and T5 to A.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815758&req=5

f1: Map of plasmid pIB11 and mutations giving rise to stimulated expression of bla. Restriction sites shown are unique. Pm, positively regulated promoter; xylS, gene encoding Pm activator; Apr (bla), ampicillin‐resistance gene encoding β‐lactamase; Kmr, kanamycin‐resistance gene; trfA, gene encoding the essential replication protein; oriV, origin of vegetative replication; oriT, origin of transfer; t, bidirectional transcriptional terminator; rrnBT1T2, bidirectional transcriptional terminator. Details for the DNA sequences corresponding to the Pm 5′‐untranslated transcript region (UTR) are displayed above the plasmid map. SD is the putative Shine–Dalgarno sequence. Nucleotides in lowercase were randomly mutagenized. Deletion mutations are indicated by short horizontal lines. Transcriptional and translational start sites are indicated with an arrow. The following base substitutions were identified in four selected examples from the screening of the LI library (nucleotide numbers are indicated in the 5′ to 3′ direction). Sequence 1: A2C, C3A, A14T. Sequence 2: C3A, A10C, T12C, A14C. Sequence 3: A2T, C3A, G6C, T12C, A14T. Sequence 4: A2T, A8G, C9T, T12C, A14T, T15A. Note that A10C is also found in nine of the 12 LII sequences and T12C is found in four. The LI library was constructed in plasmid pLB1, which differs from pIB11 in the UTR region in that A4 has been changed to T, and T5 to A.
Mentions: Initially, a mutated Pm UTR library (LI) was constructed in the corresponding part of the Pm/xylS expression cassette (bla used as reporter gene) by using randomly mutated synthetic oligonucleotides. The mutations were introduced throughout almost the entire UTR sequence and mutants displaying a 10‐fold increase in ampicillin‐resistance levels compared with the wild type, could easily be isolated from this library. DNA sequence analysis showed that they carried three to six mutations in the UTR DNA sequence, none of which affected the putative SD sequence. Four examples of such mutants are indicated in the legend to Fig. 1. Most of the identified sequences had mutations near the transcriptional start site, but as this was not always the case we wanted to specifically investigate how sequences in the internal parts of the UTR affect gene expression. We therefore prepared a new mutated Pm UTR library (LII) in plasmid pIB11 (Fig. 1), based on an oligonucleotide mixture in which only bases more than 7 bp downstream of the transcriptional start site were subjected to random sequence changes. Surprisingly, clones with equally high ampicillin‐resistance levels as in LI could be identified also from this new library. Twelve such mutants were selected for further studies, first by sequencing their UTR DNA sequences (LII‐1 to ‐12), followed by new synthesis of the corresponding oligonucleotides (both strands). These were then exchanged with the corresponding part from the wild‐type pIB11, reconfirmed by DNA sequencing, and phenotype analysed with respect to ampicillin resistance of the hosts, confirming identity with the original mutants isolated. This means that the phenotypes of the mutants are caused solely by the UTR mutations.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.

Show MeSH