title: Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins.
abstract: 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.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: C E
authors.initials: CE
authors.lastname: Pritchard
authors.forename: J A
authors.initials: JA
authors.lastname: Grasby
authors.forename: F
authors.initials: F
authors.lastname: Hamy
authors.forename: A M
authors.initials: AM
authors.lastname: Zacharek
authors.forename: M
authors.initials: M
authors.lastname: Singh
authors.forename: J
authors.initials: J
authors.lastname: Karn
authors.forename: M J
authors.initials: MJ
authors.lastname: Gait
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D015694:Gene Products, rev / Q000378:metabolism*; D015696:Gene Products, tat / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D013237:Stereoisomerism; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D015694:Gene Products, rev; D015696:Gene Products, tat; D009943:Organophosphorus Compounds; D010710:Phosphates; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; C032627:methylphosphonic acid