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RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog.

ABSTRACT: A new photocrosslinking purine analog was synthesized and evaluated as a transcription substrate for Escherichia coli RNA polymerase. This analog, 8-[(4-azidophenacyl)thio]adenosine 5'-triphosphate (8-APAS-ATP) contains an aryl azide photocrosslinking group that is attached to the ATP base via a sulfur-linked arm on the 8 position of the purine ring. This position is not involved in the normal Watson-Crick base pairing needed for specific hybridization. Although 8-APAS-ATP could not replace ATP as a substrate for transcription initiation, once stable elongation complexes were formed, 8-APAS-AMP could be site-specifically incorporated into the RNA, and this transcript could be further elongated, placing the photoreactive analog at internal positions in the RNA. Irradiation of transcription elongation complexes in which the RNA contained the analog exclusively at the 3' end of an RNA 22mer, or a 23mer with the analog 1 nt from the 3' end, produced RNA crosslinks to the RNA polymerase subunits that form the RNA 3' end binding site (beta, beta'). Both 8-APAS-AMP and the related 8-azido-AMP were subjected to conformational modeling as nucleoside monophosphates and in DNA-RNA hybrids. Surprisingly, the lowest energy conformation for 8-APAS-AMP was found to be syn, while that of 8-azido-AMP was anti, suggesting that the conformational properties and transcription substrate properties of 8-azido-ATP should be re-evaluated. Although the azide and linker together are larger in 8-APAS-ATP than in 8-N(3)-ATP, the flexibility of the linker itself allows this analog to adopt several different energetically favorable conformations, making it a good substrate for the RNA polymerase.

SUBMITTER: Costas C

PROVIDER: S-EPMC103291 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

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RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog.

Costas C C Yuriev E E Meyer K L KL Guion T S TS Hanna M M MM

Nucleic acids research 20000501 9

A new photocrosslinking purine analog was synthesized and evaluated as a transcription substrate for Escherichia coli RNA polymerase. This analog, 8-[(4-azidophenacyl)thio]adenosine 5'-triphosphate (8-APAS-ATP) contains an aryl azide photocrosslinking group that is attached to the ATP base via a sulfur-linked arm on the 8 position of the purine ring. This position is not involved in the normal Watson-Crick base pairing needed for specific hybridization. Although 8-APAS-ATP could not replace ATP ...[more]

PMID: 10756182

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Project description:DEAD-box helicases are important regulators of biomolecular condensates. However, the mechanisms through which these enzymes affect the dynamics of biomolecular condensates have not been systematically explored. Here, we demonstrate the mechanism by which mutation of a DEAD-box helicaseâ€™s catalytic core alters ribonucleoprotein condensate dynamics in the presence of ATP. Through altering RNA length within the system, we are able to attribute the altered biomolecular dynamics and material properties to physical crosslinking of RNA facilitated by the mutant helicase. These results suggest the mutant condensates approach a gel transition when RNA length is increased to lengths comparable to eukaryotic mRNA. Lastly, we show that this crosslinking effect is tunable with ATP concentration, uncovering a system whose RNA mobility and material properties vary with enzyme activity. More generally, these findings point to a fundamental mechanism for modulating condensate dynamics and emergent material properties through nonequilibrium, molecular-scale interactions.SignificanceBiomolecular condensates are membraneless organelles which organize cellular biochemistry. These structures have a diversity of material properties and dynamics which are crucial to their function. How condensate properties are determined by biomolecular interactions and enzyme activity remain open questions. DEAD-box helicases have been identified as central regulators of many protein-RNA condensates, though their specific mechanistic roles are ill-defined. In this work, we demonstrate that a DEAD-box helicase mutation crosslinks condensate RNA in an ATP-dependent fashion via protein-RNA clamping. Protein and RNA diffusion can be tuned with ATP concentration, corresponding to an order of magnitude change in condensate viscosity. These findings expand our understanding of control points for cellular biomolecular condensates that have implications for medicine and bioengineering.

Dataset Information

RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog.

Publications

RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog.

Similar Datasets

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