Polynucleotide Phosphorylase Functions as Both an Exonuclease and a Poly(A) Polymerase in Spinach Chloroplasts

Shlomit Yehudai-Resheff; Merav Hirsh; Gadi Schuster

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Polynucleotide Phosphorylase Functions as Both an Exonuclease and a Poly(A) Polymerase in Spinach Chloroplasts

机译：多核苷酸磷酸化酶在菠菜叶绿体中既作为核酸外切酶又作为聚（A）聚合酶

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The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase). In Escherichia coli,polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence. While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts. Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of P_iand ADP. As expected of a phosphorylase, P_i enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. In addition, searching the completeArabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide. These results suggest that there is no enzyme similar to E. coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.

机译：叶绿体中mRNA降解的分子机制由一系列事件组成，这些事件包括内切核酸酶裂解，向内切核酸酶裂解产物中添加富含聚（A）的序列以及多核苷酸磷酸化酶（PNPase）的外切核酸酶降解。在大肠杆菌中，聚腺苷酸化主要通过聚（A）-聚合酶（PAP）I或不存在时通过PNPase进行。在尝试通过遵循体外聚腺苷酸化活性来纯化叶绿体PAP时，发现它与PNPase共纯化，实际上无法与之分离。纯化的PNPase能够使RNA分子聚腺苷酸化，其活性类似于裂解的叶绿体。两种活性都比ATP更有效地使用ADP，并且受到茎环结构的抑制。通过控制生理上相关浓度的P _{i 和ADP，PNPase的活性直接作用于RNA降解或聚合。如预期的那样，P _{i 增强降解，而ADP抑制降解并增强聚合。此外，搜索完整的拟南芥基因组发现了几种推定的PAP，其中没有一个是典型的叶绿体转运肽。这些结果表明，没有类似于 E的酶。菠菜叶绿体中的大肠杆菌PAP I和菠菜叶绿体中RNA的聚腺苷酸化和核酸外切降解是通过一种酶PNPase进行的。}} 展开▼

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《Molecular and Cellular Biology》 |2001年第16期|共9页

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Shlomit Yehudai-Resheff; Merav Hirsh; Gadi Schuster;
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中图分类细胞生物学;

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1. Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins. [J] . Cardenas PP, Carzaniga T, Zangrossi S, Nucleic Acids Research . 2011,第21期

机译：RecN，SsbA和RecA蛋白调节单链DNA末端的多核苷酸磷酸化酶核酸外切酶和聚合酶活性。

2. Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins [J] . Esther Garcia-Tirado, Federica Briani, Gianni Dehò, Nucleic acids research . 2011,第21期

机译：RecN，SsbA和RecA蛋白调节单链DNA末端的多核苷酸磷酸化酶核酸外切酶和聚合酶活性

3. Polynucleotide phosphorylase and the archaeal exosome as poly(A)-polymerases [J] . Shimyn Slomovic, Victoria Portnoy, Shlomit Yehudai Resheff Biochimica et Biophysica Acta. Gene Regulatory Mechanisms . 2008,第4期

机译：多核苷酸磷酸化酶和古细菌外来体为聚（A）-聚合酶

4. HDX/MS of the E. coli replicative DNA polymerase identifies stabilizing interactions between the DNA sliding clamp, exonuclease and clamp loader [C] . Rafael Fernandez Leiro, Sarah L. Maslen, Meindert H. Lamers, ASMS Conference on Mass Spectrometry and Allied Topics . 2016

机译：大肠杆菌复制DNA聚合酶的HDX / MS识别DNA滑动夹，外切核酸酶和夹具装载机之间的稳定相互作用

5. Optimal type three secretion system activity of the pathogenic Yersiniae require polynucleotide phosphorylase: Mediated by its S1 RNA binding domain. [D] . Rosenzweig, Jason A. 2006

机译：致病性耶尔森氏菌的最佳三型分泌系统活性需要多核苷酸磷酸化酶：由其S1 RNA结合结构域介导。

6. Polynucleotide Phosphorylase Functions as Both an Exonuclease and a Poly(A) Polymerase in Spinach Chloroplasts [O] . Shlomit Yehudai-Resheff, Merav Hirsh, Gadi Schuster 2001

机译：多核苷酸磷酸化酶在菠菜叶绿体中既作为核酸外切酶又作为聚（A）聚合酶

7. Polynucleotide Phosphorylase Functions as Both an Exonuclease and a Poly(A) Polymerase in Spinach Chloroplasts [O] . Yehudai-Resheff, Shlomit, Hirsh, Merav, Schuster, Gadi 2001

机译：多核苷酸磷酸化酶在菠菜叶绿体中既作为核酸外切酶又作为聚（A）聚合酶

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1. 实时PCR检测中抗高温聚合酶核酸外切酶活性的荧光探针 [P] . 中国专利： CN1900312B . 2016.08.03

2. 实时PCR检测中抗高温聚合酶核酸外切酶活性的荧光探针 [P] . 中国专利： CN1900312A . 2007-01-24

3. Plasmid incorporating polynucleotide phosphorylase gene - and a DNA fragment of a bacterial plasmid, and bacterial strains contg. the plasmid used to prepare phosphorylase polynucleotide [P] . 外国专利： FR2508059B1 . 1983-11-18

机译：包含多核苷酸磷酸化酶基因的质粒-细菌质粒的DNA片段，以及细菌菌株contg。用于制备磷酸化酶多核苷酸的质粒

4. Plasmid incorporating polynucleotide phosphorylase gene - and a DNA fragment of a bacterial plasmid, and bacterial strains contg. the plasmid used to prepare phosphorylase polynucleotide [P] . 外国专利： FR2508059A1 . 1982-12-24

机译：包含多核苷酸磷酸化酶基因的质粒-细菌质粒的DNA片段，以及细菌菌株contg。用于制备磷酸化酶多核苷酸的质粒

5. Method of nucleotide identification using an off-switch through proofreading 3' exonuclease-resistant modified primers by polymerases with 3' exonuclease activity [P] . 外国专利： US2006172307A1 . 2006-08-03

机译：通过具有3'核酸外切酶活性的聚合酶对3'核酸外切酶抗性修饰引物进行校对的方法，使用开关进行核苷酸鉴定

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Polynucleotide Phosphorylase Functions as Both an Exonuclease and a Poly(A) Polymerase in Spinach Chloroplasts

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