RNA binding by the tat and rev proteins of HIV-1.
HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)
Karn
J
J
Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
Dingwall
C
C
Finch
J T
JT
Heaphy
S
S
Gait
M J
MJ
D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D015694:Gene Products, rev / Q000378:metabolism*; D015696:Gene Products, tat / Q000378:metabolism*; D015752:Genes, env; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D011533:Proviruses / Q000235:genetics; D012326:RNA Splicing; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins; D015533:Transcriptional Activation; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D002352:Carrier Proteins; D015694:Gene Products, rev; D015696:Gene Products, tat; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus