Sequences Flanking the Classical G-Box Element (CACGTG) Affect Gene Expression

Cis-elements in promoters or enhancers are major determinants of the binding specificity of transcription factors for transcriptional regulation. To improve our understanding of how these short DNA sequences affect gene expression, synthetic promoters consisting of a classical G-box core sequence (CACGTG) along with different flanking sequences were constructed and used to regulate a gfp gene. Evaluation of the effects of these flanking sequences on gene expression was performed using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Use of the classical 6-bp G-box provided a base level of gene expression while the four flanking bases on either side of the G-box were modified and the effects on gene expression were evaluated. The proximal 2-bp sequences on either flank of the G-box significantly affected the activity of G-box, while the distal 2-bp flanking nucleotides also influenced gene expression albeit with a decreasing effect. Mutagenesis of 2-4 bp flanks of the G-box showed that a G-box could drive higher gene expression only if its flanking nucleotides had widening minor groove widths (MGWs), indicating the flanking sequences of promoter elements might affect the binding specificity and intensity of transcription factors via influencing the DNA shape for protein binding. Our study could lead to a better understanding of the mechanism of cis-acting elements on gene regulation and also provide key information for precise promoter modulation in plants through targeted mutagenesis.