信號肽同步表達和易位對相鄰基因座位點運動的影響

Abstract

由於原核生物並沒有核膜將染色體包覆住，故在基因表達的過程中，染色體將有機會與細胞內膜產生互動。而關於染色體與細胞內膜互動的研究中，轉嵌現象(Transertion)是一個人們感興趣的議題。轉嵌現象意指原核生物在未有核膜阻隔下，膜蛋白基因表達時mRNA轉錄、肽鍊轉譯與蛋白質易位三件事同時發生的現象。目前已有許多間接證據說明三者同時發生的可行性，但仍無法完全證實該現象亦無法證實染色體育細胞膜間的物理連結。為了進一步證實轉嵌現象作用，我們選擇易於調控的乳糖操縱組作為膜蛋白樣品，並利用ParB-mCherry/parC2系統及TetR-YFP/tetO系統在膜蛋白上下游加入螢光標記，以觀察膜蛋白表達時對於周圍染色體基因座運動的影響。此外，我們想進一步探討不同嵌入途徑膜蛋白在轉嵌運動上的變化，故我們將乳糖操縱組中膜蛋白lacY序列替換成其他膜蛋白訊息序列進行實驗。最後，我們將膜蛋白序列與上下游標記放置於染色體不同結構域中進行觀察，以進一步估計單一膜蛋白產生之轉嵌現象對於染色體整體組織的影響。我們的結果顯示染色體在類核各處結構域中的運動皆屬於碎形布朗運動。關於嵌入途徑對轉嵌現象的影響，相當遺憾的我們並未觀察到明顯的差距，其原因可能在於我們對於膜蛋白基因的處理，僅選擇訊息序列可能並不足以產生足夠明顯的轉嵌現象。而針對不同結構域的實驗，我們發現位於類核較外側的Ori-MD相較於其他結構域可以觀察到更明顯的轉嵌現象作用變化，且同一個結構域中，不同位點之間並未觀察的明顯分類。因此我們推測對於轉嵌現象而言，其所處位置的局部結構可能會對運動影響更大，且單一膜蛋白轉嵌現象所能產生的作用有限，因此當膜蛋白位於類核內側時，轉嵌現象的結構便可能受到染色體組織限制。

In prokaryotes, the absence of a nuclear membrane enclosing the chromosomes allows for interactions between chromosomes and the cell membrane during gene expression. One topic of interest in the study of these interactions is the phenomenon of Transertion, which refers to the simultaneous occurrence of transcription, translation, and translocation during membrane protein gene expression. While numerous indirect pieces of evidence support the feasibility of these processes occurring simultaneously, definitive proof of this phenomenon and mechanical connections between chromosomes and the cell membrane remain elusive.To further validate the mechanism of Transertion, we chose the lac operon as the target membrane protein gene. Utilizing the ParB-mCherry/parC2 system and the TetR-YFP/tetO system, we introduced fluorescent markers upstream and downstream of the membrane protein to observe the influence of membrane protein expression on the motion of vicinal gene loci. Additionally, we aimed to investigate the variations in Transertion movements among different membrane protein insertion pathways by replacing the lacY gene with other membrane protein signal sequences. Finally, we placed the membrane protein gene and their fluorescent labels within different chromosomal Macrodomains to estimate the overall impact of Transertion on chromosome organization.Our results show that the motion of gene within various chromosome macrodomains follows subdiffusive fractional Brownian motion. Regarding the influence of insertion pathways on Transertion, we did not observe significant differences. This lack of distinction might be due to our approach of using only the signal sequences, which may not be sufficient to induce a pronounced Transertion phenomenon. In our experiments targeting different chromosome macrodomains, we observed more evident changes in Transertion in the Ori-MD region, located at the nucleoid periphery, compared to other domains. Furthermore, no significant categorization was observed among different loci within the same macrodomains. Thus, we hypothesize that the local structure's position may have a greater impact on Transertion, and the effect of a single membrane protein's Transertion phenomenon is limited. Consequently, when the membrane protein is located within the inner regions of the nucleoid, the structural influence of Transertion may be restricted by chromosomal organization.