Two distinct nuclear transcription factors recognize loop and bulge residues of the HIV-1 TAR RNA hairpin.
Transcriptional activation by the HIV-1 Tat protein requires specific residues in the hexanucleotide loop and trinucleotide bulge of the TAR RNA stem-loop structure found in the 5'-untranslated leader of all viral transcripts. Tat directly contacts residue U22 in the bulge and is thought to act in concert with cellular factors bound to the loop. We find that HeLa nuclear extracts contain two specific TAR RNA-binding proteins, designated TRP-1 and TRP-2, which compete for binding to the upper portion of the TAR hairpin. Analysis of point mutants in TAR RNA reveals that TRP-1 contacts residues in the loop that are important for trans-activation, whereas TRP-2 contacts the bulge, including the same residue (U22) that is required for the Tat-TAR interaction. Glycerol gradient sedimentation and UV cross-linking experiments indicate that TRP-1 is a large heteromeric complex containing a 185-kD RNA-binding protein, whereas TRP-2 activity derives from a family of 110- to 70-kD proteins. Interestingly, both TRP-1 and TRP-2 promote TAR-dependent transcription in vitro in the presence of Tat, although mixing experiments indicate that each of the three proteins must bind independently to TAR RNA. These findings suggest that the TAR element is recognized by two different nuclear RNA-binding proteins that affect transcriptional regulation by Tat.
Sheline
C T
CT
Salk Institute, La Jolla, California 92037.
Milocco
L H
LH
Jones
K A
KA
D001483:Base Sequence; D001667:Binding, Competitive; D015696:Gene Products, tat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein
D015696:Gene Products, tat; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein