• 미생물학회지
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• 제31권3호
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• pp.208-213
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• 1993

The structural and functional roles of IGS element of T4 td intron in thymidylate synthase activity in vivo were investigated Site-directed mutagenesis was employed to crete mutations of IGS element of T4 td intron, When a U-G pari was changed to a U-C pari in the 5' splice site of P1 stem of td intron, the activity of thymidylate synthase was completely abolished whereas the wild type retained the normal activity of enzyme. When U at 12 position within IGS element was changed to C, the activity of thymidylate synthase was approximately 32% of that of the wild type. Comparison of enzyme activities suggests that IGS element within P1 structure is an essential requirement for splicing of td gene in vivo.

• 미생물학회지
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• 제35권4호
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• pp.253-257
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• 1999

Cadaverine, putrescine, spermidine과 spermine이 spectinomycin에 의한 T4 파지 thymidylate synthase 유전자(td) intron의 splicing 억제에 미치는 영향을 조사하였다. Polyamine이 존재하지 않는 상태에서 7mM spectinomycin은 splicing rate를 약 40% 감소시켰다. 사용한 농도 범위(0.1~5mM)에서 cadaverine은 splicing rate를 감소시켰으나, putrescine은 0.5mM 농도에서 약 13% 정도의 splicing rate를 증가시켰다. Spermidine은 0.5mM 농도에서 약 11% 정도의 splicing rate를 증가시켰으며, sperimine은 0.01mM 농도에서 약 16% 정도의 splicing rate을 증가시켰다. 시험한 polyamine 중에서 특히 sperimine은 가장 낮은 농도에서 spectinomycin에 의한 억제반응을 극복하는 최고의 활성효과를 나타냈다. 이와 같은 억제 회복 효과는 polyamine에 의한 td intron 리보자임의 구조적 안정성에 기인하는 것으로 추정된다.

• Journal of Microbiology
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• 제33권2호
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• pp.160-164
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• 1995

The splicing activity of T4 phage td intron RNA has been examined with various Mg$^{2+}$ ions such as MGCl$_{2}$, MgS $O_{4}$ and magnesium acetate using various splicing conditions such as different incubation time and temperature. The maximum splicing of td intron RNA occurred at the concentration of 5 mM MgCl$_{2}$. Raising the Mg$^{2+}$ concentration up to 15 mM appeared to promote P2 delection mutant to overcome the loss of some splicing activity. In both wild type and mutant, a complete hydrolysis of RNA occurred at 30 mM MgCI$_{2}$ MgS $O_{4}$ and magnesium acetate exhibited the rate and pattern of RNA splicing identical to MGCI$_{2}$. The optimal splicing conditions involve the incubation of RNA with 5 mM MgCI$_{2}$ at 58 .deg.C for 15 min. The results suggest that Mg$^{2+}$ may play a key role in the catalytic mechanism of td intron RNA.n RNA.

• Animal cells and systems
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• 제4권2호
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• pp.141-144
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• 2000

Effects of deamido-$\textrm{NAD}^{+}$on self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td) was investigated. The self-splicing was not affected by deamido-$\textrm{NAD}^{+}$- at concentrations up to 2 mM. However, it began to decrease at 5 mM and the formation of splicing products such as the linear intron, intron-exon 2 and exon 1-exon 2, was slightly reduced. At 20 mM the self-splicing activity was almost completely abolished. This analog of the coenzyme $\textrm{NAD}^{+}$- inhibits the self-splicing of td intron RNA although it does not possess a guanidine group in its structure. The analysis of inhibitory concentrations and structural examination suggests that the key structural features of deamido-$\textrm{NAD}^{+}$ responsible for the inhibition of splicing may be the ADP-ribose moiety.

• Journal of Microbiology
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• 제37권4호
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• pp.243-247
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• 1999

Effects of divalent cations on self-splicing inhibition by the antibiotic spectinomycin of the phage T4 thymidylate synthase intron (td) have been investigated. $Ca^{2+}$ ion at 1mM concentration suppressed splicing inhibition of spectinomycin by 10% and 50 ${\mu}M\;Co^{2+}$ ion also suppressed splicing inhibition of specinomycin by 10%. $Mg^{2+}$ ion at 6 mM concentration decreased splicing inhibition of spectinomycin by 42% while $Mn^{2+}$ ion decreased the splicing inhibition by 10%. $Zn^{2+}$ ion at 10 uM concentration lowered the splicing inhibition by spectinomycin of 15%. Of all divalent cations tested, $Mg^{2+}$ ion was the most effective in suppressing splicing inhibition by specinomycin whereas $Ca^{2+}$ ion was the least effective. The results suggest that spectinomycin may interact with specific and functional $Mg^{2+}$-binding sites within intron RNA that lead to a displacement of $Mg^{2+}$ essential for catalytic activity.

• 미생물학회지
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• 제38권1호
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• pp.13-18
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• 2002

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 $Zn^{2+}$ 과 $Mn^{2+}$ 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.

• Journal of Microbiology
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• 제34권1호
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• pp.49-53
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• 1996

The effects of K$^{+}$ ion on the activity of RNA splicing of T4 phage thymidylate synthase gene have been investigated. The splicing activity was stimulated within the range of 5 to 20 mM concentration of KCI. When the concentration of KCI in the splicing reaction was brought to 100 or 200 mM a small amount of the exonl-intron product (1, 4 kb) was formed with large proportion of primary RNA transcript not undergoing splicing. This observation strongly suggests that there may exist come kinds of interferences with transesterification at the first step of splicing. Overall it can be concluded that K$^{+}$ ion exhibits very unique roles in RNA splicing of tdd gene depending on its concentration.ion.

• Journal of Microbiology
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• 제40권2호
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• pp.134-139
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• 2002

Effects of GTP on the inhibition of self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td) by thiamine pyrophosphate and its analogs have been investigated. The order of the inhibitory efficiency for compounds tested was as follows: thiamine pyrophosphate > thiamine monophosphate > thiamine. of all compounds examined, thiamine pyrophosphate was the most potent inhibitor, Increasing GTP concentration in splicing reaction tended to overcome the suppressive effects of self-splicing by thiamine pyrophosphate and its analogs. The inhibition by thiamine pyrophosphate was most sensitized to a higher concentration of GTP, It has been speculated that the key structural features in thiamine pyrophosphate and its analogs responsible for the inhibition of splicing may be a thiamine moiety in which the phosphorylation of 2-hydroxylethyl group on 5-position of thiazolium ring rendered further stimulation of inhibition in self-splicing reaction..