Analytical Sciences

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

Takahiko NOJIMA,*^1,*² Angela C. LIN,*¹ Teruo FUJII,*^1,*³ and Isao ENDO*^1,*⁴

*1 Biochemical Systems Laboratory, The Institute of Physical and Chemical Research (RIKEN), Saitama 351-0198, Japan
*2 Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Fukuoka 819-0395, Japan
*3 Institute of Industrial Science, The University of Tokyo, Tokyo 153-8505, Japan
*4 Saitama Industrial Technology Center, Saitama 333-0844, Japan

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.

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