Key Labeling Technologies to Tackle Sizeable Problems in RNA Structural Biology

Dayie, T. Kwaku

doi:10.3390/ijms9071214

Cited by 42 publications

(39 citation statements)

References 112 publications

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“…[6–8] However, uniform labeling introduces direct one-bond and residual dipolar 13 C- 13 C couplings that exacerbate resolution, sensitivity, and linewidth problems, and hinder accurate measurement of 13 C relaxation parameters. [9] …”

mentioning

confidence: 99%

Regio‐Selective Chemical‐Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies

et al. 2014

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Isotope labeling revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs requires site-specific labeling tools to expedite NMR structural and dynamics studies. Using enzymes from the pentose phosphate pathway, we couple chemically synthesized uracil nucleobase with specifically 13C-labeled ribose to synthesize both UTP and CTP with nearly quantitative yields. This chemo-enzymatic method affords a cost-effective preparation of labels that are unattainable by current methods. The methodology generates versatile 13C and 15N labeling patterns which, when employed with relaxation-optimized NMR spectroscopy, effectively mitigates problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and importance: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro-tRNA (76 nt), and HIV-1 core encapsidation signal (155 nt).

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confidence: 99%

Regio‐Selective Chemical‐Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies

et al. 2014

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“…Finally, while the current study used affinity chromatography for the end stage, previous studies used streptomycin sulfate to precipitate the nucleic acids, ammonium sulfate to precipitate the proteins followed by a DEAE chromatographic step and a final ammonium sulfate precipitation steps [24,26]. Affinity chromatography is known to typically produce a higher protein yield and higher protein purity, compared to ammonium sulfate precipitations and gel filtration [29]. Use of uniform expression hosts and single step affinity purification means that all six enzymes can be overexpressed and purified in two days, saving time and making the process less laborious.…”

Section: Discussionmentioning

confidence: 99%

“…This hurdle must be overcome to make this method widely adopted by the biophysics and chemical biology communities. Of these six enzymes, only ribokinase and adenosine phosphoribosyltransferase (APRT) have robust activities of 350-700 U (U is the unit of activity, defined as μmol of substrate turned over per min) per liter of bacterial culture [29]. The other five are only moderately over-expressed with activities of 28-40 U, making the enzyme preparation labor intensive [17, 24, 26].…”

Section: Introductionmentioning

confidence: 99%

Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway

Arthur

Alvarado

Dayie

2011

Protein Expression and Purification

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RNAs, more than ever before, are increasingly viewed as biomolecules of the future, in the versatility of their functions and intricate three-dimensional folding. To effectively study them by nuclear magnetic resonance (NMR) spectroscopy, structural biologists need to tackle two critical challenges of spectral overcrowding and fast signal decay for large RNAs. Stable-isotope nucleotide labeling is one attractive solution to the overlap problem. Hence, developing effective methods for nucleotide labeling is highly desirable. In this work, we have developed a facile and streamlined source of recombinant enzymes from the pentose phosphate pathway for making such labeled nucleotides. The Escherichia coli (E. coli) genes encoding ribokinase (RK), adenine phosphoribosyltranferase (APRT), xanthine/guanine phosphoribosyltransferase (XGPRT), and uracil phosphoribosyltranferase (UPRT) were subcloned into pET15b vectors. All 4 constructs together with cytidine triphosphate synthetase (CTPS) and human phosphoribosyl pyrophosphate synthetase isoform 1 (PRPPS) were transformed into the E. coli BL21 (AI) strain for protein overexpression. The enzyme preparations were purified to >90% homogeneity by a one-step Ni-NTA affinity chromatography, without the need of a further size-exclusion chromatography step. We obtained yields of 1530, 22, 482, 3120, 2120 and 2280 units of activity per liter of culture for RK, PRPPS, APRT, XGPRT, UPRT and CTPS, respectively; the specific activities were found to be 70, 22, 21, 128, 144 and 113 U/mg, respectively. These specific activities of these enzyme constructs are comparable to or higher than those previously reported. In addition, both the growth conditions and purification protocols have been streamlined so that all the recombinant proteins can be expressed, purified and characterized in at most two days. The availability and reliability of these constructs should make production of fully and site-specific labeled nucleotides for making labeled RNA accessible and straightforward, to facilitate high-resolution NMR spectroscopic and other biophysical studies.

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“…In the subsequent discussion we will focus mainly on preparation of samples of proteins and protein assemblies. There are intense developments in the sample preparation of viral DNA and RNA systems [60–62], but these will not be addressed here in any detail.…”

Section: Recent Methodological Advances For Mas Nmr Studies Of Virmentioning

confidence: 99%

Magic angle spinning NMR of viruses

Quinn

Suiter

et al. 2015

Progress in Nuclear Magnetic Resonance Spectroscopy

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Viruses, relatively simple pathogens, are able to replicate in many living organisms and to adapt to various environments. Conventional atomic-resolution structural biology techniques, X-ray crystallography and solution NMR spectroscopy provided abundant information on the structures of individual proteins and nucleic acids comprising viruses; however, viral assemblies are not amenable to analysis by these techniques because of their large size, insolubility, and inherent lack of long-range order. In this article, we review the recent advances in magic angle spinning NMR spectroscopy that enabled atomic-resolution analysis of structure and dynamics of large viral systems and give examples of several exciting case studies.

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Key Labeling Technologies to Tackle Sizeable Problems in RNA Structural Biology

Cited by 42 publications

References 112 publications

Regio‐Selective Chemical‐Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies

Regio‐Selective Chemical‐Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies

Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway

Magic angle spinning NMR of viruses

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