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Abstract Selective stable isotope labeling has transformed structural and dynamics analysis of RNA by NMR spectroscopy. These methods can remove13C-13C dipolar couplings that complicate13C relaxation analyses. While these phenomena are well documented for sites with adjacent13C nuclei (e.g. ribose C1′), less is known about so-called isolated sites (e.g. adenosine C2). To investigate and quantify the effects of long-range (> 2 Å)13C-13C dipolar interactions on RNA dynamics, we simulated adenosine C2 relaxation rates in uniformly [U-13C/15N]-ATP or selectively [2-13C]-ATP labeled RNAs. Our simulations predict non-negligible13C-13C dipolar contributions from adenosine C4, C5, and C6 to C2 longitudinal (R1) relaxation rates in [U-13C/15N]-ATP labeled RNAs. Moreover, these contributions increase at higher magnetic fields and molecular weights to introduce discrepancies that exceed 50%. This will become increasingly important at GHz fields. Experimental R1measurements in the 61 nucleotide human hepatitis B virus encapsidation signal ε RNA labeled with [U-13C/15N]-ATP or [2-13C]-ATP corroborate these simulations. Thus, in the absence of selectively labeled samples, long-range13C-13C dipolar contributions must be explicitly taken into account when interpreting adenosine C2 R1rates in terms of motional models for large RNAs.
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HPLC and 32P ‐radiolabeling method for quantification of microbial adenylate concentrations and turnover rates in seawater
Abstract We optimized a high throughput method to quantify turnover rates of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in marine microbes from simultaneous measures of the respective stocks and phosphorylation rates. We combined a microbial adenylate extraction method using boiling 20 mM Tris buffer with purification and analysis by high pressure liquid chromatography optimized to quantify these intracellular adenylate concentrations in marine microbes. Additionally, we incorporated radiolabeled phosphate (32Pi) incubations to quantify phosphorus (P) uptake rates and the phosphorylation rates for these adenylate compounds in microbial cells. With this method, we can directly assess the variations in microbial growth rates, metabolic turnover rates, energy charge, and adenylate storage. We applied and validated this method application with environmental samples from Biscayne Bay, Florida, and quantified adenylate turnover times of 12, 15, and 73 min, for ATP, ADP, and AMP, respectively. Future incorporation of this method into experiments and geographic surveys across marine environments will allow for direct assessments of changes in microbial metabolic activity in relation to other ecological variables.
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- Award ID(s):
- 2126668
- PAR ID:
- 10428726
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Limnology and Oceanography: Methods
- Volume:
- 20
- Issue:
- 8
- ISSN:
- 1541-5856
- Page Range / eLocation ID:
- p. 482-499
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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