以單分子螢光共振能量轉移技術探討核醣體蛋白bS1與rpsA 5’UTR之間的交互作用

Abstract

轉譯作用於核醣體進行，其中bS1是核醣體上分子量最大、與30S結合力最弱，且與mRNA具非專一性結合力的蛋白。實驗室先前的研究成果已知bS1會先結合於rpsO mRNA之5’UTR (非轉譯區) 雙髮夾結構間的單股序列，再解開下游hairpin 2的髮夾結構以增強結合的穩定性並促進轉譯的起始。然而bS1與它自身的mRNA-rpsA 之間是否也存在類似的結合規律，是否也具有蛋白結合的結構或者序列 偏好，尚屬未知。因此我的研究將逐步修改rpsA 5’UTR的組成結構與序列，並以單分子螢光共振能量轉移技術探討bS1蛋白是如何結合至RNA上，進而調控自身的轉譯作用。此外，由於先前的研究中發現bS1蛋白間具有協同性 (cooperativity)，且此協同性可能來自於蛋白質N端與C端之間的交互作用，因此於本研究中也使用除去D6 domain的bS1蛋白突變體bS1-DD6進行實驗，以探討兩者結合能力的差異。 我們發現當保留rpsA 5’UTR的第二與第三髮夾結構及中間的單股序列時，bS1即能夠很好地與其結合，且再進一步去掉hairpin 3後依然能使bS1良好結合，然而去除hairpin 2卻無法得到類似的結果。此外，當更換rpsA 5’UTR的第二段單股序列 (連接hairpin 2與3)為rpsO 5’UTR中相對應的單股片段時，將獲得截然不同的結果：於bS1不存在的情況下，RNA本身即具有多種動態與靜態存在的形式，而在加入bS1之後，更能觀察到複雜多樣的動態變化。對於置換單股序列造成的迥異結果，我們推論bS1蛋白可能是藉由其中特定的結構域有順序地先結合至rpsA 5’UTR的第二段單股序列，再向上游打開hairpin 2的結構，如此才能使蛋白穩定地結合。 我們也將與bS1結合良好的mRNA再與bS1-DD6進行單分子實驗，並做動力學分析，發現以單一速率常數即能夠很好地擬合蛋白的結合速率，且其與加入的蛋白濃度成正比，然而需要以兩個速率常數才能夠較好地擬合蛋白的解離速率，推測是由於多個bS1的結合以及bS1結合至RNA上不同的位置所造成的結果。此外我發現bS1-DD6能夠更快地結合至保留下游兩個髮夾結構的rpsA (rpsA-HP2-HP3)上，且其解離速率也較bS1慢。然而若再去除第三個髮夾則會觀察到bS1-DD6比bS1更快地解離，因而推論bS1的D6 domain可能會阻礙其與RNA結合，而於rpsA (rpsA-HP2-HP3)上所觀察到bS1-DD6的結合時間較長，則可能還包含bS1某些結構域與RNA間的相互影響。綜合以上，我們對於bS1與rpsA 5’UTR之間的交互作用提出新的見解。

Translation is carried out on ribosomes, among which bS1 is the protein with the largest molecular weight, the weakest binding affinity to the ribosome, and non-sequence-specificity for mRNA. The previous research results of our laboratory have shown that bS1 first binds to the single-stranded sequence between the rpsO 5’UTR double hairpin structure, and then unwinds the downstream hairpin 2 to enhance the stability of the binding and promote the initiation of translation. However, it is unknown whether a similar binding mechanism follows between bS1 and its own mRNA, rpsA, and whether it also has a structure- or sequence-binding preference. Hence, I will progressively modify the structure and sequence of the rpsA 5’UTR, and use the single-molecule fluorescence resonance energy transfer technology to explore how bS1 protein binds to it to regulate its own translation. In addition, since the cooperativity between bS1 proteins was found in previous studies and it may come from the interaction between the N-terminus and C-terminus of the protein, the bS1 protein mutant without the D6 domain (bS1-DD6) was also used in this study to explore the difference in binding ability between them. We found that when the second and third hairpin structures, together with the single-stranded linker, of rpsA 5’UTR were retained, bS1 could bind well to it. Further removal of hairpin 3 could still make bS1 bind well, but similar results were not obtained when hairpin 2 was deleted. In addition, when the single-stranded linker between hairpins 2 and 3 of the rpsA 5’UTR was replaced with the corresponding single-stranded fragment from the rpsO 5’UTR, completely different results were obtained. In the absence of bS1, RNA itself has a a variety of dynamic and static forms, and after adding bS1, more complex and diverse changes can be observed. Regarding the different results caused by the replacement of the single-stranded sequence, we deduced that the binding of bS1 protein may be sequenced by specific domains therein. It may first bind to the second single -stranded linker (SS2) of the rpsA 5’UTR, and then opens the structure of upstream hairpin 2, so that the protein can be stably bound. We also performed single-molecule experiments with bS1-DD6 for mRNA that binds well to bS1, and did kinetic analysis. We found that a single rate constant could well fit the binding rate of the protein, and it was proportional to the added protein concentration. However, two rate constants are required to fit the protein dissociation rate well, presumably due to the binding of multiple bS1 and the binding of bS1 to different positions on the RNA. In addition, I found that bS1- DD6 binds faster to two-hairpin-containing rpsA (rpsA-HP2-HP3) and dissociates at a slower rate than bS1. However, on rpsA-HP2-SS2 (without hairpin 3), it was observed that bS1-DD6 dissociates faster than bS1, so it is inferred that the D6 domain of bS1 may hinder its binding to RNA, and that the longer duration of bS1-DD6 binding to rpsA (rpsA-HP2-HP3) may also involve interactions between certain domains of bS1 and RNA. Taken together, we provide new insights into the interaction between bS1 and rpsA 5’UTR.