Mapping of nucleic acid binding in proteolytic domains of HIV-1 reverse transcriptase.
Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and its domain fragments were used to map nucleic acid binding sites within the enzyme. Discrete domain fragments were produced after the digestion of three forms of RT (p66, p66/p51 heterodimer, and p51) with V8 protease or trypsin, and the primary structure of each domain fragment was mapped by both immunoblotting and N-terminal amino acid sequence analysis. These domain fragments represent N-terminal, middle, or C-terminal regions of RT. Using Northwestern or Southwestern blotting assays, the domain fragments were evaluated for nucleic acid binding. In this technique, RT proteins are electroblotted onto the membrane and renatured after SDS-PAGE; the proteins are then probed with the primer analogues 32P-labeled d(T)16 or 32P-labeled tRNA(Lys,3). A V8 protease domain fragment spanning residues 195 to approximately 300 (p12), which was found earlier to be UV cross-linked to the primer in intact RT [Sobol et al. (1991) Biochemistry 30, 10623-10631], showed binding to both nucleic acid probes. We first localized nucleic acid binding in p66 to an N-terminal domain fragment of residues 1 approximately equal to 300. By contrast, a C-terminal domain fragment termed p30(303 approximately equal to 560) did not show nucleic acid binding. To investigate the role of the region just N-terminal to residue 303, an expression vector named pRC-35 encoding residues 273-560 was constructed.(ABSTRACT TRUNCATED AT 250 WORDS)
Kumar
A
A
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555.
Kim
H R
HR
Sobol
R W
RW
Becerra
S P
SP
Lee
B J
BJ
Hatfield
D L
DL
Suhadolnik
R J
RJ
Wilson
S H
SH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D009696:Nucleic Acids; D010446:Peptide Fragments; D012357:RNA, Transfer, Lys; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin