Charging State Analysis of Transfer RNA from an α-proteobacterium
α-变形杆菌转运RNA的装载状态分析
发布: 2020年12月05日第10卷第23期 DOI: 10.21769/BioProtoc.3834 浏览次数: 2584
评审: Alka MehraSridevi Ranganathan
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参见作者原研究论文
The authors used this protocol in:
Oct 2019
Abstract
Transfer RNA (tRNA) is an essential link between the genetic code and proteins. During the process of translation, tRNA is charged with its cognate amino acid and delivers it to the ribosome, thus serving as a substrate of protein synthesis. To analyze the charging state of a particular tRNA, total RNA is purified and analyzed on an acid-urea gel. Separated RNA is then transferred to a membrane and detected with a probe for the tRNA of interest. Here, we present an improved protocol to analyze the tRNA charging state in the α-proteobacterium Rhodopseudomonas palustris. Compared to the classical method, the RNA isolation step is optimized to suit this organism. Additionally, a non-radioactive platform is used for electrophoresis and Northern blots. This significantly reduces the time and the effort required for this protocol.
Keywords: Translation (翻译)
tRNA charging (tRNA装载)
Aminoacyl-tRNA (氨酰转移RNA)
Acid urea gel (醋酸尿素凝胶)
Rhodopseudomonas palustris (沼泽红假单胞菌)
Background
The primary function of tRNA is, with the help of other translation factors, to ensure the accurate translation of mRNA to protein. Aminoacyl-tRNAs (charged) bring amino acids to the ribosome for peptide elongation, and then the uncharged tRNAs are released. The charging state of tRNA is largely determined by the available resource (i.e., amino acids) and their consumption by the ribosome. To analyze the charging state of cellular tRNA, methods have been developed using acid-urea gels to separate the total RNA and to detect the tRNA of interest by Northern blot (Janssen et al., 2012; Bullwinkle and Ibba, 2016). Recently, we explored the relationship between tRNA charging states and survival of starving bacteria (Yin et al., 2019). The method used in that study and elaborated here, has optimized the RNA purification step to better capture the tRNA charging state in bacteria other than E. coli. It also utilizes a commercially available system to perform the analysis without using radioactive materials, which could be helpful for some research groups.
Materials and Reagents
Notes:
It is crucial to keep all the materials and reagents RNase-free for this protocol. To this end, the reagents and materials are all RNase-free, and are purchased pre-made or ready-to-dissolve in order to simplify the procedure as much as possible. Disposable RNase-free centrifuge tubes are used to make reagents whenever applicable.
Multiple kits are available for DIG reagents. Items 19-23 are listed here for readers to choose from to suit the scale required.
Centrifuge tubes, RNase free (1.5 ml, 2 ml, 15 ml and 50 ml, as needed)
QIAzol Lysis Reagent (Qiagen, catalog number: 79306 )
Chloroform, molecular biology grade
Isopropanol, molecular biology grade
Ethanol, molecular biology grade
UltraPure DNase/RNase-Free Distilled Water (Invitrogen, catalog number: 10977-023 )
TEMED (National Diagnostics, catalog number: EC-503 )
Ammonium persulfate (National Diagnostics, catalog number: EC-504 )
Urea (National Diagnostics, catalog number: EC-605 )
AccuGel 29:1 (30%) (National Diagnostics, catalog number: EC-851 )
Sodium acetate, 3 M solution, pH 4.5 (National Diagnostics, catalog number: EC-905 )
Sodium acetate, 3 M solution, pH 5.2 (National Diagnostics, catalog number: EC-906 )
EDTA, 0.5 M, pH 8.0 (Thermo Fisher Scientific, catalog number: AM9260G )
Xylene cyanol, molecular biology grade
Bromophenol blue, molecular biology grade
Tris-HCl solution, 1 M, pH 9.0 (Sigma-Aldrich, catalog number: T2819-100ML )
Amersham Hybond-XL membrane (GE Life Sciences, catalog number: 45-001-148 )
Tris-broate-EDTA (TBE) buffer, 10x (Thermo Fisher Scientific, catalog number: AM9863 )
DIG RNA labeling Mix (Sigma-Aldrich, catalog number: 11277073910 )
DIG RNA labeling kit (Roche, catalog number: 11175025910 )
DIG system (Roche, catalog number: 12039672910 )
DIG Easy Hyb (Sigma-Aldrich, catalog number: 11603558001 )
DIG Wash and Block Buffer Set (Sigma-Aldrich, catalog number: 11585762001 )
Anti-Digoxigenin-AP, Fab fragments (Sigma-Aldrich, catalog number: 11093274910 )
CDP-Star (Sigma-Aldrich, catalog number: 11685627001 )
RNase Zap (Thermo Fisher, catalog number: AM9780 )
Growth medium for R. palustris (PM medium) (see Recipes)
Acid-urea gel (see Recipes)
Sodium acetate-EDTA running buffer (see Recipes)
Sodium acetate-EDTA loading buffer (see Recipes)
Equipment
Note: The working bench as well as electrophoresis, transfer and Northern blot systems should be cleaned with RNaseZap or equivalent to decontaminate RNase before use.
Zirconia/Silica Beads, 0.1 mm dia (Bio Spec Products Inc, manufacture number: 11079101z )
Mini-Beadbeater-24 (Bio Spec Products Inc, catalog number: 112011 )
Electrophoresis, transfer and Northern blot system that fits a gel 20 cm in height
Temperature-controlled tabletop centrifuge
PCR machine for temperature-controlled incubation or equivalent incubator
Software
ImageJ
Procedure
文章信息
版权信息
© 2020 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Yin, L. and Harwood, C. S. (2020). Charging State Analysis of Transfer RNA from an α-proteobacterium. Bio-protocol 10(23): e3834. DOI: 10.21769/BioProtoc.3834.
Download Citation in RIS Format
分类
分子生物学 > RNA
微生物学 > 微生物生物化学 > RNA
微生物学 > 微生物生理学 > 适应
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