       Document 0319
 DOCN  M9610319
 TI    The amino terminal domain of HIV-1 Rev is required for discrimination of
       the RRE from nonspecific RNA.
 DT    9601
 AU    Daly TJ; Doten RC; Rusche JR; Auer M; Repligen Corporation, Cambridge,
       MA 02139, USA.
 SO    J Mol Biol. 1995 Oct 20;253(2):243-58. Unique Identifier : AIDSLINE
       MED/96036758
 AB    The ability of HIV-1 Rev to successfully discriminate between specific
       Rev-responsive elements (RRE) and nonspecific binding sites in the
       presence of excess nonspecific RNA was examined using filter binding,
       gel shift, and gel filtration techniques, using purified M4 Rev mutant
       protein and endoproteinase Lys-C cleaved wild-type Rev. The M4 Rev
       displayed a slightly reduced binding affinity to the RRE, as well as a
       tenfold decrease in its ability to discriminate the RRE from
       non-specific RNA compared to the wild-type Rev. Gel shift and gel
       filtration chromotography data also showed decreased ability of the
       mutant to multimerize in the absence or presence of the RRE. The Lys-C
       cleaved Rev, which lacks the amino-terminal 20 amino acids of the
       protein, displayed less ability to discriminate the RRE from nonspecific
       RNA compared to either the wild-type or the M4 mutant Rev and appeared
       unable to form protein-protein interactions, yet still bound sense and
       antisense RNA species with high affinity (Kd was in the nanomolar
       concentration range). A 40 amino acid peptide containing the
       arginine-rich RRE binding domain of Rev was also observed to interact
       with both the RRE and antisense RNA fragments with a binding constant of
       about 1 x 10(-9) M. However, the peptide displayed almost no ability to
       discriminate between the RRE and a comparably sized antisense RRE. The
       loss in ability to discriminate correct from incorrect binding sites
       correlates with overall decreases in the alpha-helical character of the
       protein and perturbations within the amino terminus. The amino terminus
       of Rev is likely to maintain the conformational integrity of the
       arginine rich RRE binding domain which is required for specific RNA
       binding site discrimination or stabilization of specific Rev-RRE
       interactions.
 DE    Amino Acid Sequence  Base Sequence  Binding Sites  Binding, Competitive
       Circular Dichroism  Cloning, Molecular  Comparative Study  Escherichia
       coli  Gene Products, rev/*CHEMISTRY/ISOLATION & PURIF/*METABOLISM
       HIV-1/*METABOLISM  Kinetics  Macromolecular Systems  Mathematics
       Models, Structural  Models, Theoretical  Molecular Sequence Data
       *Nucleic Acid Conformation  Peptide Fragments/CHEMISTRY/CHEMICAL
       SYNTHESIS  Recombinant Proteins/CHEMISTRY/ISOLATION & PURIF/METABOLISM
       RNA, Antisense/*CHEMISTRY/ISOLATION & PURIF/METABOLISM  RNA,
       Viral/*CHEMISTRY/ISOLATION & PURIF/METABOLISM  Substrate Specificity
       JOURNAL ARTICLE

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

