Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland.
Microb Cell Fact. 2018 Sep 21;17(1):150. doi: 10.1186/s12934-018-0999-3.
Epimutations arising from transcriptional slippage seem to have more important role in regulating gene expression than earlier though. Since the level and the fidelity of transcription primarily determine the overall efficiency of gene expression, all factors contributing to their decrease should be identified and optimized.
To examine the influence of A/T homopolymeric sequences on introduction of erroneous nucleotides by slippage mechanism green fluorescence protein (GFP) reporter was chosen. The in- or out-of-frame gfp gene was fused to upstream fragment with variable number of adenine or thymine stretches resulting in several hybrid GFP proteins with diverse amino acids at N-terminus. Here, by using T7 phage expression system we showed that the intensity of GFP fluorescence mainly depends on the number of the retained natural amino acids. While the lack of serine (S) residue results in negligible effects, the lack of serine and lysine (SK) contributed to a significant reduction in fluorescence by 2.7-fold for polyA-based in-frame controls and twofold for polyTs. What is more, N-terminal tails amino acid composition was rather of secondary importance, since the whole-cell fluorescence differed in a range of 9-18% between corresponding polyA- and polyT-based constructs.
Here we present experimental evidence for utility of GFP reporter for accurate estimation of A/T homopolymeric sequence contribution in transcriptional slippage induction. We showed that the intensity of GFP hybrid fluorescence mainly depends on the number of retained natural amino acids, thus fluorescence raw data need to be referred to appropriate positive control. Moreover, only in case of GFP hybrids with relatively short N-terminal tags the fluorescence level solely reflects production yield, what further indicates the impact of an individual slippage sequence. Our results demonstrate that in contrast to the E. coli enzyme, T7 RNA polymerase exhibits extremely high propensity to slippage even on runs as short as 3 adenine or 4 thymine residues.
转录滑动引起的表观遗传突变似乎比以前在调节基因表达方面发挥了更重要的作用。由于转录的水平和保真度主要决定了基因表达的整体效率，因此应该确定并优化所有有助于降低转录的因素。
为了研究 A/T 同聚核苷酸序列对滑动机制引入错误核苷酸的影响，选择了绿色荧光蛋白（GFP）报告基因。将框内或框外 GFP 基因与具有不同腺嘌呤或胸腺嘧啶延伸数的上游片段融合，导致 N 端具有不同氨基酸的几种杂种 GFP 蛋白。在这里，我们使用 T7 噬菌体表达系统表明 GFP 荧光的强度主要取决于保留的天然氨基酸的数量。虽然缺乏丝氨酸（S）残基的影响可以忽略不计，但缺乏丝氨酸和赖氨酸（SK）导致荧光强度显著降低，对于基于 polyA 的框内对照为 2.7 倍，对于 polyTs 为两倍。更重要的是，N 端尾部氨基酸组成是次要的，因为相应的 polyA 和 polyT 构建体之间的全细胞荧光差异在 9-18%的范围内。
在这里，我们提供了使用 GFP 报告基因准确估计转录滑动诱导中 A/T 同聚核苷酸序列贡献的实验证据。我们表明 GFP 杂种荧光的强度主要取决于保留的天然氨基酸的数量，因此荧光原始数据需要参考适当的阳性对照。此外，只有在 GFP 杂种具有相对较短的 N 端标签的情况下，荧光水平才仅反映产量，这进一步表明了单个滑动序列的影响。我们的结果表明，与大肠杆菌酶相反，T7 RNA 聚合酶甚至在短至 3 个腺嘌呤或 4 个胸腺嘧啶残基的运行中就表现出极高的滑动倾向。
