Nucleic Acids Research, Vol 27, Issue 5 1369-1376, Copyright © 1999 by Oxford University Press
MM Hanna, E Yuriev, J Zhang and DL Riggs
We report the synthesis and characterization of 5-
thioacetamidofluorescein-uridine 5'-triphosphate (5-SF-UTP), and its
application to the characterization of the environment of the nascent RNA
during trans-cription. This analog specifically replaced UTP as a
transcription substrate for Escherichia coli and T7 RNA polymerases, and
yeast RNA polymerase III. Escherichia coli transcription complexes
containing analog incorporated at only position +21 of the RNA were
prepared. The RNA was then elongated in the absence of analog, 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 relative
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 the 3'
end of the RNA, but only slightly above that of free RNA, suggesting that
the probe was on the solvent-exposed surface of the polymerase. Molecular
modeling of these analog-substituted RNAs produced structures consistent
with the experimental data. The excellent substrate properties of this
analog make it useful for the characterization of the environment of RNA
not only during transcription and translation, but in any type of
ribonucleoprotein complex.
ARTICLES
Probing the environment of nascent RNA in Escherichia coli transcription elongation complexes utilizing a new fluorescent ribonucleotide analog
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval Room 208, Norman, OK 73019-0370, USA. mhanna@chemdept.chem.ou.edu
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  C. Costas, E. Yuriev, K. L. Meyer, T. S. Guion, and M. M. Hanna RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog Nucleic Acids Res., May 1, 2000; 28(9): 1849 - 1858. [Abstract] [Full Text] [PDF]
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