Determination of the Termination Efficiency of the Transcription Terminator Using Different Fluorescent Profiles in Green Fluorescent Protein Mutants

• Published in: Analytical Sciences Vol. 21; no. 12; pp. 1479 - 1481
• Main Authors: NOJIMA, Takahiko, LIN, Angela C., FUJII, Teruo, ENDO, Isao
• Format: Journal Article
• Language: English
• Published: Japan The Japan Society for Analytical Chemistry 2005; Japan Science and Technology Agency
• Subjects: Base Sequence; DNA Primers; Escherichia coli; Escherichia coli - genetics; Green Fluorescent Proteins - genetics; Mutation; Nucleic Acid Conformation; Terminator Regions, Genetic; Transcription, Genetic
• ISSN: 0910-6340; 1348-2246
• DOI: 10.2116/analsci.21.1479

An approach in determining the intrinsic termination efficiency (%T) of transcription termination using green fluorescent protein (GFP) mutants was developed. This approach utilizes a cassette vector in which the tested terminator is introduced between two GFP mutant genes: an ultraviolet-optimized mutant (GFPuv: F99S, M153T, V163A) and a blue-shifted mutant (BFP: F64L, S65T, T145F). The ratio of the fluorescence intensity of BFP to GFPuv after transcription and translation represents the termination efficiency of the terminator. E. coli ribosomal RNA operon T1 terminator, phage lambda terminator site R2, E. coli tryptophane attenuater were introduced into the vector, and their transcriptional efficiencies were estimated as 89, 79, and 24%, respectively, showing good agreement with published data.