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1994-08-27
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Document 0704
DOCN M9480704
TI Methylphosphonate mapping of phosphate contacts critical for RNA
recognition by the human immunodeficiency virus tat and rev proteins.
DT 9410
AU Pritchard CE; Grasby JA; Hamy F; Zacharek AM; Singh M; Karn J; Gait MJ;
MRC Laboratory of Molecular Biology, Cambridge, UK.
SO Nucleic Acids Res. 1994 Jul 11;22(13):2592-600. Unique Identifier :
AIDSLINE MED/94316502
AB The HIV-1 regulatory proteins tat and rev are both RNA binding proteins
which recognize sequences in duplex RNA which are close to structural
distortions. Here we identify phosphate contacts which are critical for
each binding reaction by use of a new method. Model RNA binding sites
are constructed carrying substitutions of individual phosphodiesters by
uncharged methylphosphonate derivatives isolated separately as Rp and Sp
diastereoisomers and tested for protein binding by competition assays.
In the binding of tat to the trans-activation response region (TAR),
three phosphates, P21 and P22 which are adjacent to the U-rich bulge and
P40 on the opposite strand, are essential and in each case both isomers
inhibit binding. Similarly, in the interaction between the HIV-1 rev
protein and the rev-responsive element (RRE) both methylphosphonate
isomers at P103, P104, P124 and P125 interfere with rev binding. At
P106, only the Rp methylphosphonate isomer is impaired in rev binding
ability and it is proposed that the Rp oxygen is hydrogen-bonded to an
uncharged amino acid or to a main chain hydrogen atom. Synthetic
chemistry techniques also provide evidence for the conformations of
non-Watson-Crick G106:G129 and G105:A131 base-pairs in the RRE 'bubble'
structure upon rev binding. Almost all functional groups on the 5 bulged
residues in the bubble have been ruled out as sites of contact with rev
but, by contrast, the N7-positions of each G residue in the flanking
base-pairs are identified as sites of likely hydrogen-bonding to rev.
The results show that both tat and rev recognize the major groove of
distorted RNA helixes and that both proteins make specific contacts with
phosphates which are displaced from the sites of base-pair contact.
DE Base Composition Base Sequence Binding Sites Cloning, Molecular Gene
Products, rev/*METABOLISM Gene Products, tat/*METABOLISM
HIV-1/*METABOLISM Molecular Sequence Data Nucleic Acid Conformation
Organophosphorus Compounds/*METABOLISM Phosphates/METABOLISM RNA,
Viral/*METABOLISM Stereoisomers Support, Non-U.S. Gov't JOURNAL
ARTICLE
SOURCE: National Library of Medicine. NOTICE: This material may be
protected by Copyright Law (Title 17, U.S.Code).