Document 0217
 DOCN  M9440217
 TI    Studies on primer binding of HIV-1 reverse transcriptase using a
       fluorescent probe.
 DT    9404
 AU    Delahunty MD; Wilson SH; Karpel RL; Department of Chemistry and
       Biochemistry, University of Maryland; Baltimore County 21228.
 SO    J Mol Biol. 1994 Feb 18;236(2):469-79. Unique Identifier : AIDSLINE
       MED/94149751
 AB    The fluorescent nucleotide analog,
       2',3'-trinitrophenyladenosine-5'-triphosphate (TNP-ATP), was utilized to
       quantify the affinities of human immunodeficiency virus-1 reverse
       transcriptase (HIV-1 RT) for its substrates. Interaction of this probe
       with the enzyme brings about a twofold increase in the magnitude of
       fluorescence emission from the probe, and a blue-shift in wavelength
       maximum, from 561 to 553 nm. TNP-ATP binds HIV-1 RT with a dissociation
       constant of 21 microM. The presence of millimolar levels of
       deoxynucleoside triphosphates or micromolar levels of an oligonucleotide
       primer analogue, p(dT)12-18, suppressed this enhancement of
       fluorescence. The fact that inhibition was achieved with much lower
       levels of primer than of dNTPs suggests that TNP-ATP is a probe for the
       binding site of primer on the enzyme, rather than that of
       deoxynucleoside triphosphate. In support of this, the effect of TNP-ATP
       on the kinetics of DNA synthesis catalyzed by the enzyme indicated that
       the probe is a competitive inhibitor with respect to template-primer.
       The ability of primers and primer analogs to reverse the fluorescence
       enhancement was determined, and the corresponding affinities of these
       compounds for reverse transcriptase were calculated. The affinity
       increased with primer length, increasing more than 50-fold from a span
       of 5 to 15 nucleotide residues. The interaction of polydeoxynucleotides
       was consistent with a model in which the enzyme bound at adjacent
       internal sites of about 15 residues in length. Several mammalian and
       bacterial transfer RNA primers were tested, including the natural
       primer, tRNA(3Lys). The affinities were found to be between 0.55 and 1.2
       microM, with no obvious selectivity for the natural primer, which had a
       Kd of 0.79 microM. These results are discussed within the context of
       data for HIV-1 RT obtained by other methodologies.
 DE    Adenosine Triphosphate/ANALOGS & DERIVATIVES/METABOLISM  Fluorescent
       Dyes  HIV-1/*ENZYMOLOGY  Kinetics  Reverse Transcriptase/*METABOLISM
       RNA, Transfer, Lys/METABOLISM  Spectrometry, Fluorescence  Support,
       Non-U.S. Gov't  Support, U.S. Gov't, P.H.S.  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).