Fluorimetric analysis of recombinant p15 HIV-1 ribonuclease H.
We have exploited the sole tryptophan residue (Trp535) in the ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) to study features of the isolated polypeptide (p15 RNase H) by fluorescence spectroscopy. Incubation of purified p15 RNase H with a synthetic RNA/DNA hybrid was accompanied by an alteration in Trp535 fluorescence intensity. This property was used to determine an apparent binding constant (Kapp) of 3.5 x 10(6) M-1 for p15 RNase H complexed with poly(rA)/oligo(dT)12-18 and an occluded site size of 4 nucleotides. A cooperativity coefficient (omega) of 910 was also determined which indicated that nearly three logs of the Kapp were due to cooperativity effects. Recombinant p15 RNase H preparations containing mutations at position 478 (Glu478-->Gln478) or 539 (His539 -->Phe539), which are highly conserved between bacterial and retroviral RNases H, were also analyzed. Under the same conditions, these mutants failed to bind the RNA/DNA hybrid, although they were structurally similar to the wild type polypeptide. Fluorescence spectroscopy thus appears to be an alternative and sensitive means of analyzing functional properties of the purified RNase H domain of HIV-1 RT under a variety of conditions.
Cirino
N M
NM
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Kalayjian
R C
RC
Jentoft
J E
JE
Le Grice
S F
SF
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D017354:Point Mutation; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016914:Ribonuclease H / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012313:RNA; D014364:Tryptophan; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H