N-terminal fusion length: The key to reliable and context-preserving regulatory sequence characterization.

N-terminal fusion length significantly impacts the reliability of regulatory-sequence characterization, with fusions of 90 bp or longer showing strong correlations in E. coli.

Summary and findings

This study evaluated the impact of N-terminal fusion length (45-180 bp) on GFP reporter expression in E. coli. The findings indicated that fusions of 90 bp or longer achieved strong correlations (mean R² > 0.75) between reporter fluorescence and target protein activity. Fusions as short as 45 bp failed to rescue context-sensitive cases.

Abstract

Regulatory sequences are commonly characterized using fluorescent reporters, yet how N-terminal coding context shapes these measurements has not been systematically quantified. Here, we evaluated the impact of N-terminal fusion length (45-180 bp) from four genes (<i>lacZ</i>, <i>icd</i>, <i>zwf</i>, <i>bfp</i>) on GFP reporter expression driven by 15 different promoter-RBS combinations in <i>E. coli</i> with normalized fluorescence, enzymatic activity assays and transcription analysis for a representative subset of constructs. Our results demonstrate that N-terminal fusion critically determines the reliability of regulatory-sequence characterization in target-gene-specific coding contexts, with strong gene- and length-dependent effects. Fusions as short as 45 bp failed to rescue context-sensitive cases. However, among the tested fusion lengths, fusions of 90 bp or longer achieved strong correlations (mean R² > 0.75) between reporter fluorescence and target protein activity. Among the factors examined, N-terminal mRNA secondary structure showed a closer association with these fusion-length-dependent effects than transcription or translation initiation changes. This practical, context-preserving fusion strategy provides cost-effective guidance for scalable and accurate regulatory sequence profiling.

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