METHYLPHOSPHONATE MAPPING OF PHOSPHATE CONTACTS CRITICAL FOR RNA RECOGNITION BY THE HUMAN-IMMUNODEFICIENCY-VIRUS TAT AND REV PROTEINS

The HIV-1 regulatory proteins tat and rev are both RNA binding protein s which recognize sequences in duplex RNA which are close to structura l distortions. Here we identify phosphate contacts which are critical for each binding reaction by use of a new method. Model RNA binding si tes are constructed carrying substitutions of individual phosphodieste rs by uncharged methylphosphonate derivatives isolated separately as R p and Sp diastereoisomers and tested for protein binding by competitio n assays. In the binding of tat to the transactivation 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 th e HIV-1 rev protein and the rev-responsive element (RRE) both methylph osphonate isomers at P103, P104, P124 and P125 interfere with rev bind ing. At P106, only the Rp methylphosphonate isomer is impaired in rev binding ability and it is proposed that the Rp oxygen is hydrogen-bond ed to an uncharged amino acid or to a main chain hydrogen atom. Synthe tic chemistry techniques also provide evidence for the conformations o f non-Watson - Crick G(106):G(129) and G(105):A(131) 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 o f contact with rev but, by contrast, the N-7-positions of each G resid ue in the flanking base-pairs are identified as sites of likely hydrog en-bonding to rev. The results show that both tat and rev recognize th e major groove of distorted RNA helixes and that both proteins make sp ecific contacts with phosphates which are displaced from the sites of base-pair contact.