酵母核糖体及其相关蛋白质和mRNA的一步亲和纯化。
One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs.
作者信息
Inada Toshifumi, Winstall Eric, Tarun Salvador Z, Yates John R, Schieltz Dave, Sachs Alan B
机构信息
Department of Molecular and Cell Biology, University of California at Berkeley, 94720, USA.
出版信息
RNA. 2002 Jul;8(7):948-58. doi: 10.1017/s1355838202026018.
Abstract
We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae. Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials. The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs. Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome. In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNA's translational status within the cell. The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs.
摘要
我们描述了一种从出芽酵母酿酒酵母中纯化核糖体的一步亲和方法。仅表达C末端标记的Rpl25蛋白的酵母菌株提取物或在内源Rpl25p存在下过表达该蛋白的酵母菌株提取物用作起始材料。该纯化方法对标记的60S亚基具有特异性，并导致80S亚基和多核糖体以及核糖体相关蛋白和mRNA的共纯化。其中两种相关蛋白Mpt4p和Asc1p与核糖体的结合接近化学计量。此外，发现与纯化核糖体结合的mRNA程度反映了该mRNA在细胞内的翻译状态。从粗提物中一步纯化核糖体及其相关成分，应为核糖体未来的结构和生化研究以及翻译mRNA的表达谱分析提供重要工具。
相似文献
1
One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs.酵母核糖体及其相关蛋白质和mRNA的一步亲和纯化。
RNA. 2002 Jul;8(7):948-58. doi: 10.1017/s1355838202026018.
2
Affinity purification of ribosomes to access the translatome.通过核糖体亲和纯化来获取翻译组。
Methods. 2009 Jul;48(3):306-10. doi: 10.1016/j.ymeth.2009.04.003. Epub 2009 May 3.
3
Yeast N(alpha)-terminal acetyltransferases are associated with ribosomes.酵母N（α）-末端乙酰转移酶与核糖体相关。
J Cell Biochem. 2008 Feb 1;103(2):492-508. doi: 10.1002/jcb.21418.
4
Yeast Asc1p and mammalian RACK1 are functionally orthologous core 40S ribosomal proteins that repress gene expression.酵母Asc1p和哺乳动物RACK1是功能上直系同源的核心40S核糖体蛋白，可抑制基因表达。
Mol Cell Biol. 2004 Sep;24(18):8276-87. doi: 10.1128/MCB.24.18.8276-8287.2004.
5
Isolation of mRNAs encoding peroxisomal proteins from yeast using a combined cell fractionation and affinity purification procedure.采用细胞分级分离与亲和纯化相结合的方法从酵母中分离编码过氧化物酶体蛋白的mRNA。
Methods Mol Biol. 2011;714:323-33. doi: 10.1007/978-1-61779-005-8_20.
6
Tag-mediated isolation of yeast mitochondrial ribosome and mass spectrometric identification of its new components.标签介导的酵母线粒体核糖体分离及其新组分的质谱鉴定
Eur J Biochem. 2002 Nov;269(21):5203-14. doi: 10.1046/j.1432-1033.2002.03226.x.
7
The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm.大多数酵母UPF1与细胞质中的多核糖体共定位。
Mol Biol Cell. 1995 May;6(5):611-25. doi: 10.1091/mbc.6.5.611.
8
Sampling Saccharomyces cerevisiae cells by rapid filtration improves the yield of mRNAs.通过快速过滤对酿酒酵母细胞进行取样可提高mRNA的产量。
FEMS Yeast Res. 2004 May;4(7):751-6. doi: 10.1016/j.femsyr.2004.02.004.
9
Composition and functional characterization of yeast 66S ribosome assembly intermediates.酵母66S核糖体组装中间体的组成与功能表征
Mol Cell. 2001 Sep;8(3):505-15. doi: 10.1016/s1097-2765(01)00344-6.
10
Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.Npa1p是酿酒酵母60S核糖体亚基组装早期步骤所需的一种必需反式作用因子。
RNA. 2004 Jul;10(7):1073-83. doi: 10.1261/rna.7340404.
引用本文的文献
1
Comparative Analysis of Testicular Transcriptional and Translational Landscapes in Yak and Cattle-Yak: Implications for Hybrid Male Sterility.牦牛和犏牛睾丸转录组与翻译组景观的比较分析：对杂种雄性不育的启示
Biomolecules. 2025 Jul 25;15(8):1080. doi: 10.3390/biom15081080.
2
Single-molecule analysis of transcription activation: dynamics of SAGA coactivator recruitment.转录激活的单分子分析：SAGA 共激活因子募集的动力学
Nat Struct Mol Biol. 2025 Apr;32(4):675-686. doi: 10.1038/s41594-024-01451-y. Epub 2025 Jan 14.
3
Ribosome profiling: a powerful tool in oncological research.核糖体谱分析：肿瘤学研究中的一项强大工具。
Biomark Res. 2024 Jan 25;12(1):11. doi: 10.1186/s40364-024-00562-4.
4
Workability of mRNA Sequencing for Predicting Protein Abundance.mRNA 测序预测蛋白质丰度的可行性。
Genes (Basel). 2023 Nov 11;14(11):2065. doi: 10.3390/genes14112065.
5
Translatomics to explore dynamic differences in immunocytes in the tumor microenvironment.翻译组学用于探索肿瘤微环境中免疫细胞的动态差异。
Mol Ther Nucleic Acids. 2023 Sep 20;34:102037. doi: 10.1016/j.omtn.2023.102037. eCollection 2023 Dec 12.
6
Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development.Znf598 介导的 Rps10/eS10 泛素化有助于斑马鱼发育过程中核糖体泛素化动力学。
RNA. 2023 Dec;29(12):1910-1927. doi: 10.1261/rna.079633.123. Epub 2023 Sep 26.
7
What, where, and how: Regulation of translation and the translational landscape in plants.什么、哪里和如何：植物中转录和翻译景观的调控。
Plant Cell. 2024 May 1;36(5):1540-1564. doi: 10.1093/plcell/koad197.
8
Functional Micropeptides Encoded by Long Non-Coding RNAs: A Comprehensive Review.长链非编码RNA编码的功能性微肽：综述
Front Mol Biosci. 2022 Jun 13;9:817517. doi: 10.3389/fmolb.2022.817517. eCollection 2022.
9
Promising Assays for Examining a Putative Role of Ribosomal Heterogeneity in COVID-19 Susceptibility and Severity.用于研究核糖体异质性在新冠病毒易感性和严重性中假定作用的有前景的检测方法。
Life (Basel). 2022 Jan 28;12(2):203. doi: 10.3390/life12020203.
10
Seeking a Role for Translational Control by Alternative Polyadenylation in .探索可变聚腺苷酸化在翻译控制中的作用 。
你提供的原文似乎不完整，最后的“in.”后面应该还有具体内容。
Microorganisms. 2021 Sep 5;9(9):1885. doi: 10.3390/microorganisms9091885.
本文引用的文献
1
[Clubfoot].[马蹄内翻足]
Ned Tijdschr Geneeskd. 1963 May 11;107:845-8.
2
Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function.脆性X智力低下蛋白靶向对神经元功能很重要的G四联体mRNA。
Cell. 2001 Nov 16;107(4):489-99. doi: 10.1016/s0092-8674(01)00566-9.
3
Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome.微阵列技术鉴定脆性X综合征中与脆性X智力低下蛋白（FMRP）相关的脑信使核糖核酸（mRNAs）及改变的信使核糖核酸翻译谱
Cell. 2001 Nov 16;107(4):477-87. doi: 10.1016/s0092-8674(01)00568-2.
4
Identification of a 60S preribosomal particle that is closely linked to nuclear export.一种与核输出密切相关的60S核糖体前体颗粒的鉴定。
Mol Cell. 2001 Sep;8(3):517-29. doi: 10.1016/s1097-2765(01)00342-2.
5
Composition and functional characterization of yeast 66S ribosome assembly intermediates.酵母66S核糖体组装中间体的组成与功能表征
Mol Cell. 2001 Sep;8(3):505-15. doi: 10.1016/s1097-2765(01)00344-6.
6
Ray38p, a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, is a ribosome-associated protein and dissociated from ribosomes prior to the induction of cycloheximide resistance in Candida maltosa.Ray38p是嘌呤基序三螺旋DNA结合蛋白Stm1p的同源物，是一种核糖体相关蛋白，在麦芽糖假丝酵母中诱导环己酰亚胺抗性之前从核糖体上解离。
Biochem Biophys Res Commun. 2001 Jun 1;284(1):194-202. doi: 10.1006/bbrc.2001.4951.
7
Translation control: bridging the gap between genomics and proteomics?翻译控制：弥合基因组学与蛋白质组学之间的差距？
Trends Biochem Sci. 2001 Apr;26(4):225-9. doi: 10.1016/s0968-0004(00)01776-x.
8
Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source.酿酒酵母从可发酵碳源快速转移到不可发酵碳源时基因组反应中的全局和特定翻译调控
Mol Cell Biol. 2001 Feb;21(3):916-27. doi: 10.1128/MCB.21.3.916-927.2001.
9
Genome-wide location and function of DNA binding proteins.DNA结合蛋白的全基因组定位与功能
Science. 2000 Dec 22;290(5500):2306-9. doi: 10.1126/science.290.5500.2306.
10
Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.利用cDNA阵列鉴定信使核糖核蛋白复合物中的mRNA亚群。
Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14085-90. doi: 10.1073/pnas.97.26.14085.
Zotero 插件