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Titolo:
METHYLPHOSPHONATE MAPPING OF PHOSPHATE CONTACTS CRITICAL FOR RNA RECOGNITION BY THE HUMAN-IMMUNODEFICIENCY-VIRUS TAT AND REV PROTEINS
Autore:
PRITCHARD CE; GRASBY JA; HAMY F; ZACHAREK AM; SINGH M; KARN J; GAIT MJ;
Indirizzi:
MRC,MOLEC BIOL LAB,HILLS RD CAMBRIDGE CB2 2QH ENGLAND MRC,MOLEC BIOL LAB CAMBRIDGE CB2 2QH ENGLAND
Titolo Testata:
Nucleic acids research
fascicolo: 13, volume: 22, anno: 1994,
pagine: 2592 - 2600
SICI:
0305-1048(1994)22:13<2592:MMOPCC>2.0.ZU;2-7
Fonte:
ISI
Lingua:
ENG
Soggetto:
GENE-EXPRESSION REQUIRES; HIV-1 REV; RESPONSE ELEMENT; TRANS-ACTIVATOR; VIRION EXPRESSION; TARGET SEQUENCE; MESSENGER-RNA; MAJOR GROOVE; BINDING-SITE; TYPE-1;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
45
Recensione:
Indirizzi per estratti:
Citazione:
C.E. Pritchard et al., "METHYLPHOSPHONATE MAPPING OF PHOSPHATE CONTACTS CRITICAL FOR RNA RECOGNITION BY THE HUMAN-IMMUNODEFICIENCY-VIRUS TAT AND REV PROTEINS", Nucleic acids research, 22(13), 1994, pp. 2592-2600

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 criticalfor 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 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 caseboth 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 revbinding 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 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 of contact with rev but, by contrast, the N-7-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 ofbase-pair contact.

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Documento generato il 19/09/20 alle ore 09:26:18