Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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)
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PMID:Refolded HIV-1 tat protein protects both bulge and loop nucleotides in TAR RNA from ribonucleolytic cleavage. 186 81

The structure of the TAR RNA element transcribed at the 5' end of the R region of the human immunodeficiency virus is compared to the structure of its duplicate sequence in the 3' R region of the viral genome. Based on the 5'-TAR secondary structure already described, we assessed by RNase T1 primer extension assay the degree of similarity between the 5'-TAR and the 3'-TAR RNA secondary structures. We also analysed the influence of modifications in the flanking sequences. We show that the secondary structures of the 5'-TAR and the 3'-TAR are very similar and are not influenced by the flanking sequences.
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PMID:Similarity of the 5' and 3'-TAR secondary structures in HIV-1. 837 97

Covalent linkage of the arginine-rich fragment of the Tat protein to 1,4,7,10-tetraazacyclododecane (cyclen) results in the selective cleavage of the TAR-RNA of HIV-1 (see picture; the biotin at the 5' end acts as a label for the subsequent analysis of the cleavage fragments). The cleavage occurs at room temperature and is diminished when Eu(III) ions are present-at a concentration of about 1/10 of the concentration of the peptide-cyclen conjugate. The pH dependence indicates that two ammonium ions are responsible for the cleavage reaction. The white arrows in the schematic diagram mark the cleavage sites in RNase T1, and the black arrows the sites in the peptide-cyclen conjugate.
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PMID:Selective Cleavage of the HIV-1 TAR-RNA with a Peptide-Cyclen Conjugate. 1042 97

The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F.
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PMID:Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA. 1623 19

A ribonuclease, RNase T-tat, specifically designed to hydrolyze the TAR RNA of HIV-1 virus has been engineered. The protein was made by domain swapping the TAT peptide at the loop 3 position of ribonuclease T1. The RNase T-tat maintains a guanine-specific RNA hydrolytic activity, and characteristically displayed a specific affinity for the TAR RNA of HIV-1. In the in vitro and in vivo assays, the RNase T-tat preferentially inhibited the expression of TAR-bearing mRNA through cis-TAR targeting, suggesting that RNase T-tat may be potentially useful for the disruption of the initial stage of the transcription process of HIV-1 virus.
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PMID:Creating a ribonuclease T-tat that preferentially recognizes and hydrolyzes HIV-1 TAR RNA in vitro and in vivo. 1808 2