REFOLDED HIV-1 TAT PROTEIN PROTECTS BOTH BULGE AND LOOP NUCLEOTIDES IN TAR RNA FROM RIBONUCLEOLYTIC CLEAVAGE

被引:45
作者
HARPER, JW
LOGSDON, NJ
机构
[1] Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, One Baylor Plaza
关键词
D O I
10.1021/bi00246a026
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-mu-M) 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 [K(d)(apparent) < 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. Taken together with mutagenic studies, the data suggest that in addition to making sequence-specific interactions with bulge nucleotides, tat also interacts with components in the six-base loop but in a largely sequence-independent fashion.
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页码:8060 / 8066
页数:7
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共 37 条
[11]   HIV-1 TAT TRANS-ACTIVATION REQUIRES THE LOOP SEQUENCE WITHIN TAR [J].
FENG, S ;
HOLLAND, EC .
NATURE, 1988, 334 (6178) :165-167
[12]   TAT PROTEIN FROM HUMAN IMMUNODEFICIENCY VIRUS FORMS A METAL-LINKED DIMER [J].
FRANKEL, AD ;
BREDT, DS ;
PABO, CO .
SCIENCE, 1988, 240 (4848) :70-73
[13]   CELLULAR UPTAKE OF THE TAT PROTEIN FROM HUMAN IMMUNODEFICIENCY VIRUS [J].
FRANKEL, AD ;
PABO, CO .
CELL, 1988, 55 (06) :1189-1193
[14]   ENZYMATICALLY ACTIVE ANGIOGENIN RIBONUCLEASE-A HYBRIDS FORMED BY PEPTIDE INTERCHANGE [J].
HARPER, JW ;
AULD, DS ;
RIORDAN, JF ;
VALLEE, BL .
BIOCHEMISTRY, 1988, 27 (01) :219-226
[15]   MUTATIONAL ANALYSIS OF THE TRANS-ACTIVATION-RESPONSIVE REGION OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE-I LONG TERMINAL REPEAT [J].
HAUBER, J ;
CULLEN, BR .
JOURNAL OF VIROLOGY, 1988, 62 (03) :673-679
[16]   HIV-1 REGULATOR OF VIRION EXPRESSION (REV) PROTEIN BINDS TO AN RNA STEM-LOOP STRUCTURE LOCATED WITHIN THE REV RESPONSE ELEMENT REGION [J].
HEAPHY, S ;
DINGWALL, C ;
ERNBERG, I ;
GAIT, MJ ;
GREEN, SM ;
KARN, J ;
LOWE, AD ;
SINGH, M ;
SKINNER, MA .
CELL, 1990, 60 (04) :685-693
[17]   TRANSCRIPTION BY T7 RNA-POLYMERASE IS NOT ZINC-DEPENDENT AND IS ABOLISHED ON AMIDOMETHYLATION OF CYSTEINE-347 [J].
KING, GC ;
MARTIN, CT ;
PHAM, TT ;
COLEMAN, JE .
BIOCHEMISTRY, 1986, 25 (01) :36-40
[18]   HIV-1 TAT PROTEIN INCREASES TRANSCRIPTIONAL INITIATION AND STABILIZES ELONGATION [J].
LASPIA, MF ;
RICE, AP ;
MATHEWS, MB .
CELL, 1989, 59 (02) :283-292
[19]   IDENTIFICATION AND CHARACTERIZATION OF A HELA NUCLEAR-PROTEIN THAT SPECIFICALLY BINDS TO THE TRANS-ACTIVATION-RESPONSE (TAR) ELEMENT OF HUMAN-IMMUNODEFICIENCY-VIRUS [J].
MARCINIAK, RA ;
GARCIABLANCO, MA ;
SHARP, PA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (09) :3624-3628
[20]   HIV-1 TAT PROTEIN TRANS-ACTIVATES TRANSCRIPTION INVITRO [J].
MARCINIAK, RA ;
CALNAN, BJ ;
FRANKEL, AD ;
SHARP, PA .
CELL, 1990, 63 (04) :791-802