RNA-protein crosslinking to AMP residues at internal positions in RNA witha new photocrosslinking ATP analog

A new photocrosslinking purine analog was synthesized and evaluated as a tr anscription substrate for Escherichia coil RNA polymerase. This analog, 8-[ (4-azidophenacyl)thio]adenosine 5'-triphosphate (8-APAS-ATP) contains an ar yl azide photocrosslinking group that is attached to the ATP base via a sul fur-linked arm on the 8 position of the purine ring. This position is not i nvolved in the normal Watson-Crick base pairing needed for specific hybridi zation. Although 8-APAS-ATP could not replace ATP as a substrate for transc ription initiation, once stable elongation complexes were formed, 8-APAS-AM P could be site-specifically incorporated into the RNA, and this transcript could be further elongated, placing the photoreactive analog at internal p ositions in the RNA. Irradiation of transcription elongation complexes in w hich 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 confo rmational modeling as nucleoside monophosphates and in DNA-RNA hybrids, Sur prisingly, the lowest energy conformation for 8-APAS-AMP was found to be sy n, while that of 8-azido-AMP was anti, suggesting that the conformational p roperties and transcription substrate properties of 8-azido-ATP should be r e-evaluated. Although the azide and linker together are larger in 8-APAS-AT P than in 8-N-3-ATP, the flexibility of the linker itself allows this analo g to adopt several different energetically favorable conformations, making it a good substrate for the RNA polymerase.