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Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences.
Biochemistry 1993; 32(35):9115-24B

Abstract

The compatible plasmids pKGP1-1 and pCM-X# will confer chloramphenicol resistance to Escherichia coli harboring the two plasmids if the T7 RNA polymerase produced from pKGP1-1 can recognize the T7 promoter carried on pCM-X# and transcribe the CAT gene that is cloned behind the promoter [Ikeda et al. (1992) Biochemistry 31, 9073-9080]. When E. coli harbor pKGP1-1 and a pCM-X# plasmid that carries a point mutation in the T7 promoter that destroys promoter activity (an inactive pCM-X#), the T7 RNA polymerase will not utilize the T7 promoter point mutant, will not produce CAT, and will not induce chloramphenicol resistance. The selection of mutants of T7 RNA polymerase that exhibit altered promoter recognition was pursued by randomly mutagenizing pKGP1-1 with aqueous hydroxylamine, cotransforming E. coli with the mutagenized pKGP1-1 and a mixture of seven different inactive pCM-X# plasmids, and isolating and characterizing the RNA polymerase that was present in those colonies that exhibited chloramphenicol resistance. It was established that E. coli harboring the mutant plasmid pKGP-HA1mut4 and an inactive pCM-X# are chloramphenicol-resistant and that the mutation responsible for the expression of CAT from the inactive pCM-X# plasmid is a G to A transition at nucleotide 664 of T7 gene 1 that converts glutamic acid (222) to lysine. Apparently this mutation expands the range of T7 promoter sequences that can be utilized by the enzyme. The mutant T7 RNA polymerase, GP1(Lys222), utilizes all seven inactive T7 promoter point mutants more efficiently than wild-type T7 RNA polymerase both in vivo and in vitro. Furthermore, the correlation of in vivo and in vitro promoter utilization suggests that the restoration of chloramphenicol resistance in the cotransformed E. coli results from the ability of GP1(Lys222) to initiate transcription from T7 promoter point mutants that are normally inactive.

Authors+Show Affiliations

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400.No affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8369283

Citation

Ikeda, R A., et al. "Selection and Characterization of a Mutant T7 RNA Polymerase That Recognizes an Expanded Range of T7 Promoter-like Sequences." Biochemistry, vol. 32, no. 35, 1993, pp. 9115-24.
Ikeda RA, Chang LL, Warshamana GS. Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences. Biochemistry. 1993;32(35):9115-24.
Ikeda, R. A., Chang, L. L., & Warshamana, G. S. (1993). Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences. Biochemistry, 32(35), pp. 9115-24.
Ikeda RA, Chang LL, Warshamana GS. Selection and Characterization of a Mutant T7 RNA Polymerase That Recognizes an Expanded Range of T7 Promoter-like Sequences. Biochemistry. 1993 Sep 7;32(35):9115-24. PubMed PMID: 8369283.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences. AU - Ikeda,R A, AU - Chang,L L, AU - Warshamana,G S, PY - 1993/9/7/pubmed PY - 1993/9/7/medline PY - 1993/9/7/entrez SP - 9115 EP - 24 JF - Biochemistry JO - Biochemistry VL - 32 IS - 35 N2 - The compatible plasmids pKGP1-1 and pCM-X# will confer chloramphenicol resistance to Escherichia coli harboring the two plasmids if the T7 RNA polymerase produced from pKGP1-1 can recognize the T7 promoter carried on pCM-X# and transcribe the CAT gene that is cloned behind the promoter [Ikeda et al. (1992) Biochemistry 31, 9073-9080]. When E. coli harbor pKGP1-1 and a pCM-X# plasmid that carries a point mutation in the T7 promoter that destroys promoter activity (an inactive pCM-X#), the T7 RNA polymerase will not utilize the T7 promoter point mutant, will not produce CAT, and will not induce chloramphenicol resistance. The selection of mutants of T7 RNA polymerase that exhibit altered promoter recognition was pursued by randomly mutagenizing pKGP1-1 with aqueous hydroxylamine, cotransforming E. coli with the mutagenized pKGP1-1 and a mixture of seven different inactive pCM-X# plasmids, and isolating and characterizing the RNA polymerase that was present in those colonies that exhibited chloramphenicol resistance. It was established that E. coli harboring the mutant plasmid pKGP-HA1mut4 and an inactive pCM-X# are chloramphenicol-resistant and that the mutation responsible for the expression of CAT from the inactive pCM-X# plasmid is a G to A transition at nucleotide 664 of T7 gene 1 that converts glutamic acid (222) to lysine. Apparently this mutation expands the range of T7 promoter sequences that can be utilized by the enzyme. The mutant T7 RNA polymerase, GP1(Lys222), utilizes all seven inactive T7 promoter point mutants more efficiently than wild-type T7 RNA polymerase both in vivo and in vitro. Furthermore, the correlation of in vivo and in vitro promoter utilization suggests that the restoration of chloramphenicol resistance in the cotransformed E. coli results from the ability of GP1(Lys222) to initiate transcription from T7 promoter point mutants that are normally inactive. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8369283/Selection_and_characterization_of_a_mutant_T7_RNA_polymerase_that_recognizes_an_expanded_range_of_T7_promoter_like_sequences_ L2 - https://www.lens.org/lens/search?q=citation_id:8369283 DB - PRIME DP - Unbound Medicine ER -