Nucleic Acids Research, 2000, Vol. 28, No. 9 1849-1858
© 2000 Oxford University Press
RNA–protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog
Designer Genes, Inc., 8281 East Evans Road, Suite 104, Scottsdale, AZ 85260, USA and 1Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA
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 (ß,ß'). 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-N3-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.
* To whom correspondence should be addressed. Tel: +1 480 596 9312; Fax: +1 480 991 4419; Email: mmh@designergenesinc.com Present addresses: Celina Costas, Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, University of Santiago, 15706-Santiago de Compostela, Spain Elizabeth Yuriev, Protein Crystallography, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA
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