Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus type 1 Tat protein.
The human immunodeficiency virus type 1 Tat protein binds to an RNA stem-loop structure called TAR which is present at the 5' end of all human immunodeficiency virus type 1 transcripts. This binding is centered on a bulge within the stem of TAR and is an essential step in the trans-activation process which results in a dramatic increase in viral gene expression. By analysis of a series of TAR derivatives produced by transcription or direct chemical synthesis, we determined the structural and chemical requirements for Tat binding. Tat binds well to structures which have a bulge of two to at least five unpaired bases bounded on both sides by a double-stranded RNA stem. This apparent flexibility in bulge size is in contrast to an absolute requirement for an unpaired uridine (U) in the 5'-most position of the bulge (+23). Substitution of the U with either natural bases or chemical analogs demonstrated that the imido group at the N-3 position and, possibly, the carbonyl group at the C-4 position of U are critical for Tat binding. Cytosine (C), which differs from U at only these positions, is not an acceptable substitute. Furthermore, methylation at N-3 abolishes binding. While methylation of U at the C-5 position has little effect on binding, fluorination reduces it, possibly because of its effects on relative tautomer stability at the N-3 and C-4 positions. Thus, we have identified key moieties in the U residue that are of importance for the binding of Tat to TAR RNA. We hypothesize that the invariant U is involved in hydrogen bond interactions with either another part of TAR or the TAR-binding domain in Tat.
Sumner-Smith
M
M
Allelix Biopharmaceuticals Inc., Mississauga, Ontario, Canada.
Roy
S
S
Barnett
R
R
Reid
L S
LS
Kuperman
R
R
Delling
U
U
Sonenberg
N
N
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D003596:Cytosine; D015696:Gene Products, tat / Q000378:metabolism*; D005814:Genes, Viral; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014498:Uracil; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D014498:Uracil; D003596:Cytosine