Refolded HIV-1 tat protein protects both bulge and loop nucleotides in TAR RNA from ribonucleolytic cleavage.
Substantial evidence indicates that HIV-1 trans-activation by tat protein is mediated through the TAR RNA element. This RNA forms a stem-loop structure containing a three-nucleotide bulge and a six-nucleotide loop. Previous mutagenic analysis of TAR indicates that the bulge residues and a 4 bp segment of the stem constitute, in part, the tat binding site. However, there appears to be no sequence-specific contribution of the six-base loop. We have employed a ribonuclease protection technique to explore the interaction of tat with single-stranded regions of TAR. The results indicate that tat interacts with both the bulge and loop regions of TAR. Treatment of TAR RNA with RNase A results in cleavage at U23 and U31, located in the bulge and loop regions, respectively. High concentrations (approximately 2 microM) of Escherichia coli derived tat protein, prepared by standard procedures, gave complete protection of TAR RNA from RNase A cleavage. However, under these conditions, truncated TAR derivatives in which no stem-loop structure is expected to form were also protected, indicating nonspecific binding. In order to obtain a tat preparation with enhanced specificity toward TAR RNA, methods were developed for refolding the recombinant protein. This treatment enhanced the affinity of tat for TAR by approximately 30-fold [Kd(apparent) less than 25 nM] and markedly increased its specificity for the TAR. Again, tat protected TAR RNA from RNase A cleavage at both U23 and U31. Protection was also observed with RNase T1 which cleaves TAR RNA at three G residues in the six-base loop.(ABSTRACT TRUNCATED AT 250 WORDS)
Harper
J W
JW
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Logsdon
N J
NJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein
D002352:Carrier Proteins; D015696:Gene Products, tat; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012260:Ribonucleases; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein