Effects of Divalent Cations on the Self-splicing Inhibition of Group I Intron by the Coen-zyme Thiamine Pyrophosphate

2가 양이온이 Thiamine Pyrophosphate에 의한 Group I Intron Ribozyme의 Splicing 억제에 미치는 영향

  • Published : 2002.03.01

Abstract

Effects of divalent cations such as $Mg^{2+}$, $Mn^{2+}$ and $Zn^{2+}$ on the self-splicing inhibition of the T4 phage thymidylate synthase (td) intron by the coenzyme thiamine pyrophosphate have been investigated. The splicing activity increased in proportion to the concentration of $Mg^{2+}$ up to 30 mM. Without $Mg^{2+}$in the splicing reaction the $Zn^{2+}$ ion tested in the range of 0.1-6 mM concentration only produced the splicing activity about 20% that of the normal splicing rate. A majority of the splicing products were I-E2 and E2 but El-E2 ligation product, Cl and Ll were not detected. Similar patterns of splicing products were also observed with $Mn^{2+}$. At 6 mM $Zn^{2+}$the intron RNA was hydrolyzed. $Mn^{2+}$produced a little higher splicing activity than that of $Zn^{2+}$over the range of concentrations used and at 8 mM about 28% splicing activity was observed. In contrast, $Mn^{2+}\;and\;Zn^{2+}$ ions promoted the splicing activity about 35-40% on an average in the presence of 10 mM $Mg^{2+}$. Of all divalent cations tested, $Mg^{2+}$exhibited the maximum activation effect to counteract the splicing inhibition by thiamine pyrophosphate. This appears to be due to the stabilizing effect of td intron ribozyme structure essential for the catalytic function by $Mg^{2+}$.

2가 양이온($Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$)이 조효소 thiamine pyrophosphate에 의한 T4파지 티민생합성 유전자 (td) 인트론 RNA의 splicing에 미치는 영향을 조사하였다. $Mg^{2+}$를 30 mM까지 증가시켰을 때 splicing 활성은 농도에 비례하여 증가하였다. 그러나 $Mg^{2+}$이 존재하지 않는 상태에서 0.1-4 mM 농도에 걸쳐 $Zn^{2+}$를 사용한 결과 약 20% 정도의 splicing이 일어났다. 이때 대부분의 splicing product는 인트론-엑손2 및 엑손2였고 엑손1-엑손2는 검출되지 않았다. 그리고 4 mM 농도에서는 RNA가 대부분 가수분해되는 현상이 나타났다. $Mn^{2+}$ 이온은 사용한 농도 범위 (0.1-8 mM)에서 $Zn^{2+}$ 보다는 전반적으로 약간 증가된 splicing 활성을 보였으며 8 mM 농도에서도 약 30% 정도의 splicing 활성을 보였다. $Zn^{2+}$ 이온처럼 splicing product는 인트론-엑손2 및 엑손2였고 엑손1-엑손2는 검출되지 않았 다. 반면에 splicing반응에 10 mM $Mg^{2+}$ 를 첨가했을 때 $Zn^{2+}$$Mn^{2+}$ 이온은 평균 약 35-40% 정도 splicing활성을 촉진시키는 것으로 나타났다. 실험한 2가 양이온 중에서 특히 $Mg^{2+}$ 은 가장 낮은 농도에서 thiamine pyrophosphate 에 의한 억제반응을 극복하는 최고의 활성효과를 나타냈다. 이와 같은 억제 회복 효과는 $Mg^{2+}$에 의한 리보자임의 td intron 구조적 안정성에 기인하는 것으로 추정된다.

Keywords

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