大鼠核糖体RNA一个新的结构域以及辛纳毒蛋白A链缺失突变的研究
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摘要
本论文包括三部分内容：
第一部分 裸子植物侧柏种子中一种RNA水解酶型核糖体失活蛋白(Orientin)的发现及其在大鼠核糖体RNA上作用位点的确定
核糖体失活蛋白是一类来自于细菌、真菌和高等植物的核毒素，它们通过作用于核糖体的大亚基28S RNA或23S RNA失活核糖体，从而抑制蛋白质的合成。本工作首次在裸子植物侧柏种子中发现一种新型的RNA水解酶活性的核糖体失活蛋白Orientin，它可以作用于大鼠核糖体RNA产生一个特异RNA条带。经过RNA酶法测序、引物延伸法测序、RT-PCR、两向薄层层析等方法，我们确定了Orientin是通过水解大鼠核糖体28S RNA第4451位胞苷与4452位腺苷之间的磷酸二酯键来影响核糖体的功能。Orientin在大鼠核糖体28S RNA上的作用位点所在的RNA序列在高等真核生物28S rRNA中保守性较强，推测该RNA区域参与真核核糖体催化肽链的延伸过程；同时Orientin的发现也为研究核糖体RNA中一个新的功能结构域提供了工具。大鼠核糖体28S RNA内一个磷酸二酯键的断裂造成核糖体功能的降低为支持核糖体中RNA是主要的功能分子提供了新的依据。
第二部分 核糖体失活蛋白Cinnamomin A链N端和C端区域缺失突变与酶活性关系的研究
辛纳毒蛋白（Cinnamomin）是一种来源于香樟树种子的II型核糖体失活蛋白，它的A链具有RNA N-糖苷酶活力，可以特异性水解大鼠核糖体28S RNA S/R茎环结构中A4324位的糖苷键，失活核糖体，使蛋白合成终止于肽链延伸步骤。辛纳毒蛋白A链还可以作用于超螺旋DNA分子，产生缺刻和线状DNA分子。本工作构建了辛纳毒蛋白A链的缺失突变体（D3、D5、D3-5和DC3-5），分别或同时缺失了A链C末端51个氨基酸残基、N末端52个氨基酸残基，其中突变体DC3-5是在突变体D3-5的C末端增加三个氨基酸：Ile、Cys、Arg，并在E.coli中以包涵体的形式高效表达了辛纳毒蛋白A链和它的四个缺失突变体。利用尿素变性、快速稀释法复性和DEAE-Sepharose柱层析，从纯化的包涵体中获得了一系列重组蛋白。通过蛋白质体外翻译抑制试验分析和RNA N-糖苷酶活力检测，发现与复性的重组野生型辛纳毒蛋白A链相比，四个缺失突变体都不具有明显的失活核糖体的RNA N-糖苷酶活力；同时发现缺失突变体也丧失了作用于超螺旋DNA分子，产生缺刻和线状DNA分子的活性。说明辛纳毒蛋白A链的C末端51个氨基酸残基和N末端52个氨基酸残基是维持该蛋白酶活性所必需。进一步检测发现，天然和
重组辛纳毒蛋白A链都可以在超螺旋DNA分子上产生多个脱腺嘌呤位点。推测辛纳毒蛋白A链之所以可以作用于超螺旋DNA分子，产生缺刻和线状DNA分子是由于在DNA分子上脱腺嘌呤的结果，而不是外源核酸酶污染或辛纳毒蛋白A链另外具备的超螺旋DNA分子依赖的DNase的缘故。
第三部分 酸性条件下核糖体失活蛋白Gelonin对于Sarcin/Ricin结构域RNA(SRD RNA)非特异性脱腺嘌呤的研究
I型核糖体失活蛋白Gelonin，可以特异性水解大鼠核糖体28S RNA S/R结构域中A4324的糖苷键，不可逆抑制核糖体催化的蛋白质合成。但是近来发现Gelonin可以水解已除去蛋白质的核糖体RNA和DNA分子上的糖苷键，释放腺嘌呤碱基，关于Gelonin的底物位点专一性是否改变及如何改变尚不清楚。本文利用体外合成的大鼠核糖体28S RNA Sarcin/Ricin结构域的小分子模拟物SRD RNA及其突变体为底物，研究反应体系中pH的变化对Gelonin催化脱腺嘌呤反应特异性的影响，发现在酸性条件下Gelonin可以催化SRD RNA及其突变体(包括置换突变体UUUU和缺失突变体G(GA)上非特异性脱腺嘌呤反应的发生；同时，Gelonin还可以催化不具备二级结构的poly(A)分子上的脱腺嘌呤反应，其活力随着pH值的降低而增强。通过不同pH条件下的圆二色分析发现Gelonin蛋白自身的构象并未发生明显的变化。推测酸性反应条件可能影响底物RNA分子的高级结构或质子化程度，导致非特异性脱嘌呤反应的发生。中性条件下Gelonin特异性水解核糖体28S RNA S/R结构域中A4324的糖苷键可能是由于S/R结构域特定的空间结构使A4324的糖苷键恰巧暴露于Gelonin的活性中心。
This dissertation consists of three parts:
Part ⅠCleavage-site of eukaryotic ribosomal RNA by a novel ribosome-inactivating protein
From the seeds of Biota orientalis was isolated a novel ribosome-inactivating protein (RIP) - orientin that was capable of inactivating rat ribosome and of inhibiting in vitro protein synthesis by a way different from the RNA N-glycosidase. As a specific ribonuclease, orientin cleaved only a single phosphodiester bond between the cytidine residue at position 4451 and the adenosine residue at position 4452 in rat 28S ribosomal RNA and hence produced a RNA fragment that is composed of 333 ribonucleotides at the 3' end of rat 28S ribosomal RNA. The 5'-terminus of the fragment was identified to be a hydroxyl. The sequence around the cleavage-site was highly conserved in certain higher eukaryotes. The domain that contained the cleavage-site by this novel RIP was important for the ribosomal function in protein synthesis. Also this novel RIP could be useful for probing the structure of a new specific functional domain RNA in the ribosome.
Part Ⅱ Both the N-terminal and C-terminal regions are crucial for cinnamomin A-chain to deadenylate ribosomal RNA and supercoiled double-stranded DNA
Cinnamomin is a type II ribosome-inactivating protein and its A-chain exhibits RNA N-glycosidase activity to remove an adenine in the conserved Sarcin/Ricin loop of the largest RNA in ribosome, arresting protein synthesis at the elongation step. Cinnamomin A-chain can also cleave supercoiled circular double-stranded DNA into the nicked and linear forms. The recombinant cinnamomin A-chain and its four mutants with the N-terminal 52 amino acids residues or/and the C-terminal 51 amino acids residues deleted have been expressed in E coli and purified after refolding. Recombinant cinnamomin A-chain, which exhibited the RNA N-glycosidase activity, could also release adenines from the supercoiled double-stranded DNA, whereas the deletion mutants could deadenylate neither ribosomal RNA nor supercoiled DNA. Additionally, unlike the recombinant cinnamomin A-chain, the deletion mutants could not cleave supercoiled DNA into nicked and linear forms. Taken together, these results revealed that both the N-terminal and the C-terminal regions are required for cinnamomin A-chain to depurinate ribosomal RNA and supercoiled double-stranded
DNA, and that the activity to cleave supercoiled DNA is an intrinsic property of cinnamomin A-chain.
Part Ⅲ Nonspecific deadenylation on Sarcin/Ricin Domain RNA catalyzed by Gelonin under acidic conditions
Gelonin is a single-chain ribosome-inactivating protein (RIP) that can hydrolyze the glycosidic bond of a highly conserved adenosine residue in the sarcin/ricin domain (SRD) of the largest RNA in ribosome and thus irreversibly inhibit protein synthesis. Recently, the specificity in substrate recognition was challenged by the fact that gelonin could remove adenines from some other oligoribonucleotide substrates. However, the site-specificity of gelonin to deadenylate various substrates were unknown. Hereby, the effect of pH values upon site-specificity of the deadenylation activity of gelonin was studied using the synthetic oligoribonucleotide (named SRD RNA) that mimicked the rat ribosomal sarcin/ricin domain RNA. Interestingly, gelonin gradually acquired the ability to nonspecifically remove adenines from SRD RNA when pH values changed from neutral to acidic condition. Another two SRD RNA mutants, either with the conserved adenosine deleted or with the tetraloop converted, showed very similar cleavage pattern to wild type SRD RNA, underscoring the important role of pH value in site-specificity of recognition by gelonin. Furthermore, the RNA N-glycosidase activity of gelonin was also enhanced with the decreasing of pH values. In addition, no obvious change was observed in the molecular conformation of gelonin at various pH values. Taken together, our data implied that the protonation of adenosines in SRD RNA was potentially an important factor for the non-specific
第一部分 裸子植物侧柏种子中一种RNA水解酶型核糖体失活蛋白(Orientin)的发现及其在大鼠核糖体RNA上作用位点的确定
核糖体失活蛋白是一类来自于细菌、真菌和高等植物的核毒素，它们通过作用于核糖体的大亚基28S RNA或23S RNA失活核糖体，从而抑制蛋白质的合成。本工作首次在裸子植物侧柏种子中发现一种新型的RNA水解酶活性的核糖体失活蛋白Orientin，它可以作用于大鼠核糖体RNA产生一个特异RNA条带。经过RNA酶法测序、引物延伸法测序、RT-PCR、两向薄层层析等方法，我们确定了Orientin是通过水解大鼠核糖体28S RNA第4451位胞苷与4452位腺苷之间的磷酸二酯键来影响核糖体的功能。Orientin在大鼠核糖体28S RNA上的作用位点所在的RNA序列在高等真核生物28S rRNA中保守性较强，推测该RNA区域参与真核核糖体催化肽链的延伸过程；同时Orientin的发现也为研究核糖体RNA中一个新的功能结构域提供了工具。大鼠核糖体28S RNA内一个磷酸二酯键的断裂造成核糖体功能的降低为支持核糖体中RNA是主要的功能分子提供了新的依据。
第二部分 核糖体失活蛋白Cinnamomin A链N端和C端区域缺失突变与酶活性关系的研究
辛纳毒蛋白（Cinnamomin）是一种来源于香樟树种子的II型核糖体失活蛋白，它的A链具有RNA N-糖苷酶活力，可以特异性水解大鼠核糖体28S RNA S/R茎环结构中A4324位的糖苷键，失活核糖体，使蛋白合成终止于肽链延伸步骤。辛纳毒蛋白A链还可以作用于超螺旋DNA分子，产生缺刻和线状DNA分子。本工作构建了辛纳毒蛋白A链的缺失突变体（D3、D5、D3-5和DC3-5），分别或同时缺失了A链C末端51个氨基酸残基、N末端52个氨基酸残基，其中突变体DC3-5是在突变体D3-5的C末端增加三个氨基酸：Ile、Cys、Arg，并在E.coli中以包涵体的形式高效表达了辛纳毒蛋白A链和它的四个缺失突变体。利用尿素变性、快速稀释法复性和DEAE-Sepharose柱层析，从纯化的包涵体中获得了一系列重组蛋白。通过蛋白质体外翻译抑制试验分析和RNA N-糖苷酶活力检测，发现与复性的重组野生型辛纳毒蛋白A链相比，四个缺失突变体都不具有明显的失活核糖体的RNA N-糖苷酶活力；同时发现缺失突变体也丧失了作用于超螺旋DNA分子，产生缺刻和线状DNA分子的活性。说明辛纳毒蛋白A链的C末端51个氨基酸残基和N末端52个氨基酸残基是维持该蛋白酶活性所必需。进一步检测发现，天然和
重组辛纳毒蛋白A链都可以在超螺旋DNA分子上产生多个脱腺嘌呤位点。推测辛纳毒蛋白A链之所以可以作用于超螺旋DNA分子，产生缺刻和线状DNA分子是由于在DNA分子上脱腺嘌呤的结果，而不是外源核酸酶污染或辛纳毒蛋白A链另外具备的超螺旋DNA分子依赖的DNase的缘故。
第三部分 酸性条件下核糖体失活蛋白Gelonin对于Sarcin/Ricin结构域RNA(SRD RNA)非特异性脱腺嘌呤的研究
I型核糖体失活蛋白Gelonin，可以特异性水解大鼠核糖体28S RNA S/R结构域中A4324的糖苷键，不可逆抑制核糖体催化的蛋白质合成。但是近来发现Gelonin可以水解已除去蛋白质的核糖体RNA和DNA分子上的糖苷键，释放腺嘌呤碱基，关于Gelonin的底物位点专一性是否改变及如何改变尚不清楚。本文利用体外合成的大鼠核糖体28S RNA Sarcin/Ricin结构域的小分子模拟物SRD RNA及其突变体为底物，研究反应体系中pH的变化对Gelonin催化脱腺嘌呤反应特异性的影响，发现在酸性条件下Gelonin可以催化SRD RNA及其突变体(包括置换突变体UUUU和缺失突变体G(GA)上非特异性脱腺嘌呤反应的发生；同时，Gelonin还可以催化不具备二级结构的poly(A)分子上的脱腺嘌呤反应，其活力随着pH值的降低而增强。通过不同pH条件下的圆二色分析发现Gelonin蛋白自身的构象并未发生明显的变化。推测酸性反应条件可能影响底物RNA分子的高级结构或质子化程度，导致非特异性脱嘌呤反应的发生。中性条件下Gelonin特异性水解核糖体28S RNA S/R结构域中A4324的糖苷键可能是由于S/R结构域特定的空间结构使A4324的糖苷键恰巧暴露于Gelonin的活性中心。
This dissertation consists of three parts:
Part ⅠCleavage-site of eukaryotic ribosomal RNA by a novel ribosome-inactivating protein
From the seeds of Biota orientalis was isolated a novel ribosome-inactivating protein (RIP) - orientin that was capable of inactivating rat ribosome and of inhibiting in vitro protein synthesis by a way different from the RNA N-glycosidase. As a specific ribonuclease, orientin cleaved only a single phosphodiester bond between the cytidine residue at position 4451 and the adenosine residue at position 4452 in rat 28S ribosomal RNA and hence produced a RNA fragment that is composed of 333 ribonucleotides at the 3' end of rat 28S ribosomal RNA. The 5'-terminus of the fragment was identified to be a hydroxyl. The sequence around the cleavage-site was highly conserved in certain higher eukaryotes. The domain that contained the cleavage-site by this novel RIP was important for the ribosomal function in protein synthesis. Also this novel RIP could be useful for probing the structure of a new specific functional domain RNA in the ribosome.
Part Ⅱ Both the N-terminal and C-terminal regions are crucial for cinnamomin A-chain to deadenylate ribosomal RNA and supercoiled double-stranded DNA
Cinnamomin is a type II ribosome-inactivating protein and its A-chain exhibits RNA N-glycosidase activity to remove an adenine in the conserved Sarcin/Ricin loop of the largest RNA in ribosome, arresting protein synthesis at the elongation step. Cinnamomin A-chain can also cleave supercoiled circular double-stranded DNA into the nicked and linear forms. The recombinant cinnamomin A-chain and its four mutants with the N-terminal 52 amino acids residues or/and the C-terminal 51 amino acids residues deleted have been expressed in E coli and purified after refolding. Recombinant cinnamomin A-chain, which exhibited the RNA N-glycosidase activity, could also release adenines from the supercoiled double-stranded DNA, whereas the deletion mutants could deadenylate neither ribosomal RNA nor supercoiled DNA. Additionally, unlike the recombinant cinnamomin A-chain, the deletion mutants could not cleave supercoiled DNA into nicked and linear forms. Taken together, these results revealed that both the N-terminal and the C-terminal regions are required for cinnamomin A-chain to depurinate ribosomal RNA and supercoiled double-stranded
DNA, and that the activity to cleave supercoiled DNA is an intrinsic property of cinnamomin A-chain.
Part Ⅲ Nonspecific deadenylation on Sarcin/Ricin Domain RNA catalyzed by Gelonin under acidic conditions
Gelonin is a single-chain ribosome-inactivating protein (RIP) that can hydrolyze the glycosidic bond of a highly conserved adenosine residue in the sarcin/ricin domain (SRD) of the largest RNA in ribosome and thus irreversibly inhibit protein synthesis. Recently, the specificity in substrate recognition was challenged by the fact that gelonin could remove adenines from some other oligoribonucleotide substrates. However, the site-specificity of gelonin to deadenylate various substrates were unknown. Hereby, the effect of pH values upon site-specificity of the deadenylation activity of gelonin was studied using the synthetic oligoribonucleotide (named SRD RNA) that mimicked the rat ribosomal sarcin/ricin domain RNA. Interestingly, gelonin gradually acquired the ability to nonspecifically remove adenines from SRD RNA when pH values changed from neutral to acidic condition. Another two SRD RNA mutants, either with the conserved adenosine deleted or with the tetraloop converted, showed very similar cleavage pattern to wild type SRD RNA, underscoring the important role of pH value in site-specificity of recognition by gelonin. Furthermore, the RNA N-glycosidase activity of gelonin was also enhanced with the decreasing of pH values. In addition, no obvious change was observed in the molecular conformation of gelonin at various pH values. Taken together, our data implied that the protonation of adenosines in SRD RNA was potentially an important factor for the non-specific
引文
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