Probing the environment of nascent RNA in Escherichia coli transcription elongation complexes utilizing a new fluorescent ribonucleotide analog

We report the synthesis and characterization of 5-thioacetamidofluorescein- uridine 5'-triphosphate (5-SF-UTP), and its application to the characteriza tion of the environment of the nascent RNA during transcription, This analo g specifically replaced UTP as a transcription substrate for Escherichia co li and T7 RNA polymerases, and yeast RNA polymerase ill. Escherichia coli t ranscription complexes containing analog incorporated at only position +21 of the RNA were prepared. The RNA was then elongated in the absence of anal og, moving the fluorescent group further away from the enzyme active site, and the fluorescence polarization was measured. Analog positioned near the 3' end of the transcript exhibited significantly increased polarization rel ative to that of free probe, consistent with the constrained environment of the RNA in the DNA-RNA hybrid. Analog positioned 14 nucleotides from the 3 ' end exhibited significantly decreased polarization relative to that at th e 3' end of the RNA, but only slightly above that of free RNA, suggesting t hat the probe was on the solvent-exposed surface of the polymerase, Molecul ar modeling of these analog-substituted RNAs produced structures consistent with the experimental data. The excellent substrate properties of this ana log make it useful for the characterization of the environment of RNA not o nly during transcription and translation, but in any type of ribonucleoprot ein complex.