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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used gel electrophoresis to show that the pyrimidine bulge of the HIV-1 TAR sequence causes a local bending of the helical axis. The TAR bulge caused a retardation in electrophoretic mobility in polyacrylamide gels. When this was placed adjacent to an additional bulged sequence in a linear RNA fragment, the mobility of the molecule varied sinusoidally with the spacing between the two bulges. Electrophoretic mobilities suggested that the TAR sequence context of the pyrimidine bulge causes a greater degree of axial kinking than in an equivalent randomly chosen sequence. Experiments in which an A5 bulge was progressively opposed by adenine bases inserted in the opposite strand showed that even a single opposed adenine markedly reduced electrophoretic mobility, i.e. axial bending, and two adenine bases reduced the mobility virtually to that of a normal duplex. We suggest that the pronounced kinking resulting from an unopposed bulge provides a particularly recognizable feature in RNA, and that this is the basis of the interaction between the HIV Tat protein and the TAR sequence.
J Mol Biol 1992 Jul 20
PMID:Kinking of RNA helices by bulged bases, and the structure of the human immunodeficiency virus transactivator response element. 164 Apr 50

In this study we have defined the in vitro requirements for transcriptional regulation of the HIV-2 LTR in response to the HIV-1 and HIV-2 Tat proteins and addressed potential mechanisms of Tat function. HIV-2 contains a duplicated TAR RNA stem-loop structure in contrast to the single stem-loop structure found in HIV-1 TAR RNA. We demonstrated that the HIV-2 proximal TAR RNA stem-loop structure was more important for in vitro transcriptional activation by the HIV-1 and HIV-2 Tat proteins than the distal TAR RNA stem-loop though this downstream TAR element itself was able to confer Tat-responsiveness. The role of the two HIV-2 TAR RNA stem-loop bulge sequences was less critical than the loop sequences for in vitro transcriptional activation by Tat. In addition, we demonstrated that replacing the HIV-2 TATA element with that of HIV-1 markedly reduced the overall level of Tat activation. The role of the Tat-1 and Tat-2 proteins on the synthesis of HIV-1 and HIV-2 promoter proximal and promoter distal transcripts was then investigated. In contrast to the HIV-1 promoter, the HIV-2 promoter generated abundant levels of short transcripts in vitro transcription assays likely due to the structure of its duplicated TAR element. Both Tat-1 and Tat-2 increased the level of transcripts extending to the end of the HIV-1 and HIV-2 TAR elements as well as the level of transcripts extending more than 500 nucleotides from the transcription initiation site. However, the synthesis of transcripts within 30 nucleotides of the HIV-2 LTR transcription initiation site was unchanged in either the presence or absence of Tat while the level of transcripts extending increasing distances from the HIV-2 LTR transcription initiation site were progressively stimulated in the presence of Tat. Though the HIV-1 Tat protein was a stronger inducer of HIV-1 LTR transcription than the HIV-2 Tat protein, we did not detect differences in the binding of these proteins to the HIV-1 and HIV-2 TAR RNAs. This suggested that differences in their transactivation properties may be due to alterations in their association with RNA polymerase II or associated elongation factors. (ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Biol 1995 Dec 01
PMID:Tat functions to stimulate the elongation properties of transcription complexes paused by the duplicated TAR RNA element of human immunodeficiency virus 2. 749 Jul 54

Tat strongly activates transcription of the HIV-1 provirus by stimulating both initiation and elongation. This transactivator binds to the TAR RNA element, but can also associate with cellular transcription factors, interacting with upstream promoter sequences. To achieve a better understanding of the role of Tat in the assembly of the transcriptional initiation complex in the living cell, we have examined how the activity of this protein is modified when the general transcription factor involved in the first step of this process, TBP, is overexpressed. The activity of Tat, either wild-type or fused to the DNA binding domain of GAL4 (GBTat), was tested using reporter constructs containing GAL4 binding sites upstream of a minimal promoter corresponding to the HIV-1 TATA box, with or without the TAR element. We found that overexpression of TBP led to a dramatic increase in the activity of the GBTat protein. In order to activate GBTat, TBP must be able to interact with the TATA box. Analysis of several Tat mutants indicated that both the cysteine-rich and the core domains of this transactivator are necessary and sufficient to activate transcription when TBP is overexpressed. In vitro experiments showed that Tat binds specifically to TBP. There was a correlation between the ability of different Tat mutants to bind TBP and their capacity to activate transcription in vivo. With the natural HIV-1 promoter, overexpression of TBP first stimulated and then suppressed the Tat-induced activity. This inhibition was abrogated by an increase in the intracellular levels of Tat. These experimental data indicate that Tat stimulates initiation of transcription by interacting with TBP in vivo.
J Mol Biol 1995 Jul 07
PMID:Evidence for functional interaction between the HIV-1 Tat transactivator and the TATA box binding protein in vivo. 760 68

Tat, the transactivator protein encoded by HIV-1, acts in vivo to increase transcriptional initiation and stabilize elongation. We examined the effects of purified, bacterially-expressed Tat on HIV-1 transcription in a cell-free system. Tat specifically stimulated HIV-directed transcription 12-fold in HeLa cell nuclear extracts and this effect was principally due to increased transcriptional elongation. The degree of transactivation was greatest at later times during the transcription reaction when basal levels of transcription were reduced. At early times, the proportion of basal transcriptional complexes that elongate efficiently was high. Ongoing transcription increased the number of complexes requiring Tat for efficient elongation, possibly due to the activation of a repressor(s). To examine this hypothesis, the effects of the detergent Sarkosyl on HIV transcription were studied. Sarkosyl stimulated HIV-1 transcription to a level similar to that occurring in the presence of Tat alone by improving elongation. Transcription was elevated by Sarkosyl at concentrations inhibitory to reinitiation indicating that inefficient elongation is due to transcriptional pausing. Transcriptional stimulation by Sarkosyl was a general phenomenon as it was also observed with heterologous eukaryotic promoters. Tat was capable of stimulating elongation from a heterologous promoter when Tat binding was provided by a downstream TAR element. We propose that Tat acts as a general transcription factor whose binding at the promoter overcomes inefficient transcriptional elongation.
J Mol Biol 1993 Aug 05
PMID:HIV-1 Tat overcomes inefficient transcriptional elongation in vitro. 768 12

Tat (trans-activator) proteins are early RNA binding proteins regulating lentiviral transcription. These proteins are necessary components in the life cycle of all known lentiviruses, such as the human immunodeficiency viruses (HIV) or the equine infectious anemia virus (EIAV). Tat proteins are thus ideal targets for drugs intervening with lentiviral growth. The consensus RNA binding motif (TAR, trans-activation responsive element) of HIV-1 is well characterized. Structural features of the 86 amino acid HIV-1, Zaire 2 isolate (HV1Z2) Tat protein in solution were determined by two dimensional (2D) nuclear magnetic resonance (NMR) methods and molecular dynamics (MD) calculations. In general, sequence regions corresponded to structural domains of the protein. It exhibited a hydrophobic core of 16 amino acids and a glutamine-rich domain of 17 amino acids. Part of the NH2 terminus, Val4 to Pro14, was sandwiched between these domains. Two highly flexible domains corresponded to a cysteine-rich and a basic sequence region. The 16 amino acid sequence of the core region is strictly conserved among the known Tat proteins, and the three-dimensional fold of these amino acids of HV1Z2 Tat protein was highly similar to the structure of the corresponding EIAV Tat domain. HV1Z2 Tat protein contained a well defined COOH-terminal Arg-Gly-Asp (RGD) loop similar to the recently determined decorsin RGD loop.
J Mol Biol 1995 Apr 07
PMID:Structural studies of HIV-1 Tat protein. 772 10

When tethered to heterologous DNA both Tat and VP16 can activate transcription from the HIV-1 LTR. To determine if they act by similar mechanisms, we constructed several hybrid effectors between Tat or VP16 and DNA-binding domains of GAL4 or LexA proteins. We tested these effectors on substituted reporter targets, which contained one to six GAL4 or LexA DNA-binding sites placed upstream of the HIV-1 promoter. Whereas Tat acted very inefficiently via DNA even with five DNA-binding sites, effects of VP16 were observed with a single DNA-binding site and increased with increasing number of sites. More importantly, effects of VP16 via DNA were synergistic with those of Tat via TAR RNA when both proteins were expressed simultaneously. We next created a tripartite fusion protein, which contained the GAL4 DNA-binding domain and activation domains of both Tat and VP16, which could be targeted to the HIV-1 LTR either via DNA or RNA. By introducing individual deleterious mutations into either Tat or VP16, we confirmed that effects of VP16 predominated via DNA whereas Tat but not VP16 acted via TAR RNA. Thus, Tat and VP16 act at different steps of the transcription process and increase expression from the HIV-1 LTR by different mechanisms.
J Mol Biol 1993 Dec 05
PMID:Synergism between Tat and VP16 in trans-activation of HIV-1 LTR. 825 63

We present a model for the three-dimensional structure of the HIV TAR stem-loop, based on a modeling algorithm which makes use of the known X-ray coordinates of tRNAs to generate a model structure, which has then been tested experimentally in solution by enzymatic and chemical structure probing of ribo-oligonucleotides encompassing the TAR sequence. The modeling suggested that the structure of TAR was similar to that of the anti-codon loop of tRNA(Asp), having a loop of just three single-stranded residues with a mismatched adenine excluded from the helical stem on the 3' side of the loop. The structural probing is consistent with such a structure for the loop, and reveals an unusual structure around the 5' uridine-rich bulge, which is the binding target for the transactivator protein Tat. These data may be useful in understanding the interaction of TAR with the Tat protein and may aid in the design of anti-AIDS drugs. The coordinates of the model are available on request.
J Mol Graph 1993 Jun
PMID:Modeling and solution structure probing of the HIV-1 TAR stem-loop. 834 68

The Human Immunodeficiency Virus (HIV) integrates into host cellular DNA as a double strand DNA molecule. Here a previously studied HIV isolate was examined for binding and cleavage by topoisomerase II in vitro within the 5' LTR region and human flanking DNA. A cluster of strong binding and cleavage sites in the human sequences was located approximately 850 bp upstream from the integration site. This region maps to a locus consisting of a complex repeating element, and alternating purine/pyrimidine sequences. Topoisomerase II binding and cleavage sites were also located within the HIV 5' LTR, in particular a site overlying the DNA sequence coding for TAR, another inverted repeat element in the DNA.
J Mol Biol 1993 Aug 20
PMID:A cluster of strong topoisomerase II cleavage sites is located near an integrated human immunodeficiency virus. 839 47

The inducer of short transcripts, or IST, is an unusual transcriptional element located downstream of the human immunodeficiency virus type 1 (HIV-1) promoter. IST activates HIV-1 transcription, but the resulting RNAs are short and end at approximately position +59. IST, therefore, appears to promote the formation of transcription complexes that are unable to elongate efficiently. This activity contrasts with that of TAR, the target for Tat trans-activation, which upon binding of the viral protein Tat promotes the formation of transcription complexes capable of efficient elongation through the entire viral genome. We have localized and characterized the IST element. Our results indicate that IST is located mainly between positions -5 and +26, although the sequences from positions +40 to +59 also contribute to IST activity. Unlike TAR, which is an RNA element, IST appears to be a DNA element. Thus, the HIV-1 R region is a complex regulatory region with RNA and DNA elements that promote the formation of transcription complexes with different elongation properties.
Mol Cell Biol 1993 Feb
PMID:Characterization of the inducer of short transcripts, a human immunodeficiency virus type 1 transcriptional element that activates the synthesis of short RNAs. 842 90

The 5' region of HIV-1 RNA contains functional elements involved in key steps of the retroviral cycle, such as genomic RNA transcription, splicing, translation, dimerization or initiation of reverse transcription. In the present work, we investigated the conformation of the first 500 nucleotides covering the RNA leader and the 5' gag coding sequences of HIV-MAL, using chemical probing. We provide detailed information on almost each nucleotide at one of their Watson-Crick positions and on position N-7 of purines. Experiments were conducted on two in vitro transcribed RNA fragments (1 to 707 and 1 to 311). A secondary structure model was derived by combining the experimental data, computer predictions and sequence comparison. Under conditions favoring dimerization (high salt concentration), HIV-1 RNA folds into independent structural domains that can be related to defined functional regions. The first domain corresponds to TAR forming a stable stem-loop. Intrinsic structural features are found to stabilize the TAR hairpin loop. The second domain (nucleotides 56 to 299) contains the PBS sequence, which is located in a stable subdomain constrained by a four stem junction (nucleotides 139 to 218). Although the MAL isolate has an insertion near the PBS, probably resulting from the duplication of a 23-nucleotide sequence, the structural organization of this subdomain is conserved in all other HIV-1 isolates. The third domain (nucleotides 300 to 404) contains the splice donor site, packaging and dimerization elements and the AUG initiation codon of gag. A major result is the structural versatility of this region. Two mutually exclusive structures, both equally in agreement with probing data, could modulate the different functions involving this domain. The reduced accessibility of the gag translational initiation site possibly accounts for the low efficiency of the in vitro translation of the dimer. Finally, the 5' gag coding sequences form a metastable domain.
J Mol Biol 1993 Jan 20
PMID:Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains. 842 53


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