Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a reconstituted system which models the events associated with human immunodeficiency virus type 1 (HIV-1) plus-strand transfer. These events include synthesis of plus-strand strong-stop DNA [(+) SSDNA] from a minus-strand DNA donor template covalently attached to human tRNA3Lys, tRNA primer removal, and annealing of (+) SSDNA to the minus-strand DNA acceptor template. Termination of (+) SSDNA synthesis at the methyl A (nucleotide 58) near the 3' end of tRNA3Lys reconstitutes the 18-nucleotide primer binding site (PBS). Analysis of (+) SSDNA synthesis in vitro and in HIV-1 endogenous reactions indicated another major termination site: the pseudouridine at nucleotide 55. In certain HIV-1 strains, complementarity between nucleotides 56 to 58 and the first three bases downstream of the PBS could allow all of the (+) SSDNA products to be productively transferred. Undermodification of the tRNA may be responsible for termination beyond the methyl A. In studies of tRNA removal, we find that initial cleavage of the 3' rA by RNase H is not sufficient to achieve successful strand transfer. The RNA-DNA hybrid formed by the penultimate 17 bases of tRNA still annealed to (+) SSDNA must also be destabilized. This can occur by removal of additional 3'-terminal bases by RNase H (added either in cis or trans). Alternatively, the nucleic acid chaperone activity of nucleocapsid protein (NC) can catalyze this destabilization. NC stimulates annealing of the complementary PBS sequences in (+) SSDNA and the acceptor DNA template. Reverse transcriptase also promotes annealing but to a lesser extent than NC.
...
PMID:Molecular requirements for human immunodeficiency virus type 1 plus-strand transfer: analysis in reconstituted and endogenous reverse transcription systems. 1023 40

An early step in the life cycle of the human immunodeficiency virus type 1 (HIV-1) is reverse transcription of viral RNA into proviral DNA, which can then be integrated into the host cell genome. Reverse transcription is a discontinuous process carried out by the viral encoded reverse transcriptase that displays DNA polymerase activities on RNA and DNA templates as well as an RNase H activity that degrades transcribed RNA. DNA synthesis is initiated by cellular tRNALys3 that binds at its 3'-terminus to the complementary primer binding site of the genomic RNA. The initiation of reverse transcription is itself a complex reaction that requires tRNA placement onto viral RNA and the formation of a specific primer/template complex that is recognized by reverse transcriptase. After initiation takes place, the enzyme translocates from the initially bound RNA/RNA duplex into chimeric replication intermediates and finally accommodates newly synthesized DNA/RNA hybrids. This review focuses on structure-function relationships among these various molecules that are involved in the initiation of HIV-1 reverse transcription.
...
PMID:HIV-1 reverse transcription: a brief overview focused on structure-function relationships among molecules involved in initiation of the reaction. 1032 13

Retroviral reverse transcriptase (RT) enzymes are responsible for transcribing viral RNA into double-stranded DNA. An in vitro assay to analyze the second strand transfer event during human immunodeficiency virus type 1 (HIV-1) reverse transcription has been developed. Model substrates were constructed which mimic the viral intermediate found during plus-strand strong-stop synthesis. Utilizing wild-type HIV-1 RT and a mutant E478Q RT, the requirement for RNase H activity in this strand transfer event was analyzed. In the presence of Mg2+, HIV-1 RT was able to fully support the second strand transfer reaction in vitro. However, in the presence of Mg2+, the E478Q RT mutant had no detectable RNase H activity and was unable to support strand transfer. In the presence of Mn2+, the E478Q RT yields the initial endoribonucleolytic cleavage at the penultimate C residue of the tRNA primer yet does not support strand transfer. This suggests that subsequent degradation of the RNA primer by the RNase H domain was required for strand transfer. In reactions in which the E478Q RT was complemented with exogenous RNase H enzymes, strand transfer was supported. Additionally, we have shown that HIV-1 RT is capable of supporting strand transfer with substrates that mimic tRNAHis as well as the authentic tRNA3Lys.
...
PMID:RNase H requirements for the second strand transfer reaction of human immunodeficiency virus type 1 reverse transcription. 1040 Jul 54

The Osvaldo retrotransposon has shown a high transposition rate in some strains of Drosophila buzzatii and in hybrids between D. buzzatii and its sibling D. koepferae. In order to understand the molecular basis of this phenomenon, we developed a procedure to clone a recently transposed copy with the aim of characterizing an active, full- length Osvaldo element. The complete nucleotide sequence of Osvaldo, obtained from a recent insertion site, was determined. Osvaldo is 9,045 bp long and is composed of a central coding region flanked by identical long terminal repeats (LTRs) of 1,196 bp each. Sequences homologous to the polypurine tract and tRNA-primer-binding site of retroviruses are located adjacent to the 3' and 5' LTRs, respectively. The internal region of Osvaldo contains three long open reading frames (ORFs 1, 2, and 3), comparable in size and location to gag, pol, and env retroviral genes. The conceptual translation of Osvaldo ORF1 exhibits sequence homology to HIV1 and SIV capsid (p24) and nucleocapsid (p7) mature proteins. ORF2 encodes the putative protease (PR), reverse transcriptase/ribonuclease H (RT/RH), integrase (IN), and a significant portion of the surface envelope (ENV) protein that is interrupted by a putative intron. A third ORF encodes the remaining part of the ENV protein. The predicted 62-kDa ENV protein shares several general features with membrane glycoproteins, including a potential signal peptide, a transmembrane domain near the C-terminus that could function as a membrane anchor, four consensus N-linked glycosylation motifs, and, finally, a potential protease cleavage site. The phylogenetic relationships of Osvaldo are explored, and they suggest that Osvaldo may constitute a new family of retroviruses in insects, distantly related to the previously described group of gypsy retroviruses.
...
PMID:The retrotransposon Osvaldo from Drosophila buzzatii displays all structural features of a functional retrovirus. 1040 8

The antitumor antibiotic sparsomycin is a universal and potent inhibitor of peptide bond formation and selectively acts on several human tumors. It binds to the ribosome strongly, at an unknown site, in the presence of an N-blocked donor tRNA substrate, which it stabilizes on the ribosome. Its site of action was investigated by inducing a crosslink between sparsomycin and bacterial, archaeal, and eukaryotic ribosomes complexed with P-site-bound tRNA, on irradiating with low energy ultraviolet light (at 365 nm). The crosslink was localized exclusively to the universally conserved nucleotide A2602 within the peptidyl transferase loop region of 23S-like rRNA by using a combination of a primer extension approach, RNase H fragment analysis, and crosslinking with radioactive [(125)I]phenol-alanine-sparsomycin. Crosslinking of several sparsomycin derivatives, modified near the sulfoxy group, implicated the modified uracil residue in the rRNA crosslink. The yield of the antibiotic crosslink was weak in the presence of deacylated tRNA and strong in the presence of an N-blocked P-site-bound tRNA, which, as was shown earlier, increases the accessibility of A2602 on the ribosome. We infer that both A2602 and its induced conformational switch are critically important both for the peptidyl transfer reaction and for antibiotic inhibition. This supposition is reinforced by the observation that other antibiotics that can prevent peptide bond formation in vitro inhibit, to different degrees, formation of the crosslink.
...
PMID:Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes. 1043 Aug 85

Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.
...
PMID:Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences. 1062 92

An early step in the life cycle of human immunodeficiency virus type 1 is the reverse transcription of the viral RNA genome into double-stranded DNA, which is subsequently translocated to the cell nucleus. It is then integrated into host DNA and serves as a template for viral gene expression. Reverse transcription is catalyzed by the viral enzyme reverse transcriptase and is a complex process comprising a series of RNA-dependent DNA polymerization, DNA-dependent DNA polymerization, and RNase H reactions. Strand transfer reactions are required to complete the process. Reverse transcription is initiated when a molecule of host cell tRNA(lys3), which serves as a primer, is bound to the primer binding site of viral genomic RNA. The viral nucleocapsid protein is involved in each of the initiation of reverse transcription and in subsequent strand transfer or template-switching events. We review the interactions among reverse transcriptase, viral genomic RNA, the tRNA primer of reverse transcription, and viral nucleocapsid protein in the various steps of reverse transcription, including primer placement, initiation, and processive synthesis.
...
PMID:Interactions between human immunodeficiency virus type 1 reverse transcriptase, tRNA primer, and nucleocapsid protein during reverse transcription. 1077 3

The selective packaging of the primer tRNA(Lys3) into HIV-1 particles is dependent upon the viral incorporation of the Pr160gag-pol precursor protein. In order to map a tRNA(Lys3) binding site within this precursor, we have studied the effects of mutations in Pr160gag-pol upon the selective incorporation of tRNA(Lys3). Many of these mutations were placed in a protease-negative HIV-1 proviral DNA to prevent viral protease degradation of the mutant Gag-Pol protein. C-terminal deletions of protease-negative Gag-Pol that removed the entire integrase sequence and the RNase H and connection subdomains of reverse transcriptase did not inhibit the incorporation of either the truncated Gag-Pol or the tRNA(Lys3), indicating that these regions are not required for tRNA(Lys3) binding. On the other hand, larger C-terminal deletions, which also remove the thumb subdomain sequence, did prevent tRNA(Lys3) packaging, without inhibiting viral incorporation of the truncated Gag-Pol, indicating a possible interaction between thumb subdomain sequences and tRNA(Lys3). While point mutations K249E, K249Q, and R307E in the primer grip region of the thumb subdomain have been reported to inhibit the in vitro interaction of mature reverse transcriptase with the anticodon loop of tRNA(Lys3), we find that these mutations do not inhibit tRNA(Lys3) packaging into the virus, which supports other work indicating that the anticodon loop of tRNA(Lys3) is not involved in interactions with Pr160gag-pol during tRNA(Lys3) packaging.
...
PMID:Sequences within Pr160gag-pol affecting the selective packaging of primer tRNA(Lys3) into HIV-1. 1086 Jul 20

Reverse transcription of human immunodeficiency virus type-1 is primed by cellular tRNA(Lys3), which is selectively packaged into viral particles where it is bound at its 3' terminus to a complementary sequence of viral RNA termed the primer binding site (PBS). Since cellular tRNA(Lys3) is highly conserved, it might conceivably serve as a good target for novel antagonists to block reverse transcriptase (RT) activity. In this study, we have examined a number of antisense oligodeoxyribonucleotides (ODNs) that are complementary to different parts of the tRNA primer and, therefore, may interfere with the initiation of RT-mediated DNA synthesis. We found that the stability of complexes between synthetic tRNA(Lys3 )and ODNs was significantly increased when binding occurred via sequences involved in tertiary interactions of the tRNA. In particular, ODNs with complementarity to both the variable and TPsiC stem-loop of tRNA(Lys3 )bound with high affinity to both free tRNA(Lys3 )as well as to the binary tRNA(Lys3)/RNA complex. As a result, the initiation of DNA synthesis was severely compromised under these conditions. Moreover, RT-associated RNase H activity recognized the tRNA within this ternary tRNA(Lys3)/RNA/ODN complex as an RNA template and initiated its degradation. Both this RNase H degradation of tRNA(Lys3 )as well as the altered structure of the tRNA/RNA complex, due to the binding of the ODN, contributed to the inhibition of synthesis of viral DNA. The initiation of RT activity was almost completely blocked when using ODNs that interfered with intermolecular tRNA/RNA interactions that involved both the PBS and sequences outside the PBS. Similar findings were obtained with natural preparation of tRNA(Lys3).
...
PMID:Human immunodeficiency virus type-1 reverse transcription can be inhibited in vitro by oligonucleotides that target both natural and synthetic tRNA primers. 1093 21

Truncated tRNA-DNA mimics were examined in an in vitro assay for second-strand transfer during human immunodeficiency virus type 1 (HIV-1) reverse transcription. Strand transfer in this system requires the progressive degradation of the RNA within the 18-mer tRNA-DNA (plus-strand strong stop DNA) intermediate to products approximately 8 nucleotides in length. The ability of the truncated substrates to substitute for directional processing by RNase H or reverse transcriptase (RT) was examined. Using wild-type HIV-1 RT, substrates which truncated the 5' end of the tRNA primer by 6, 9, and 12 nucleotides (Delta6, Delta9, and Delta12, respectively) were recognized by RNase H and resulted in strand transfer. An overlap of 5 nucleotides between the acceptor and newly synthesized DNA template was sufficient for strand transfer. The mutant RT, E478Q correctly catalyzed the initial cleavage of the 18-mer tRNA-DNA mimic in the presence of Mn(2+); however, no directional processing was observed. In contrast, no RNase H activity was observed with the Delta6, Delta9, and Delta12 substrates with E478Q RT in this strand transfer assay. However, when complemented with Escherichia coli RNase H, E478Q RT supported strand transfer with the truncated substrates. E478Q RT did cleave the truncated forms of the substrates, Delta6, Delta9, and Delta12, in a polymerase-independent assay. The size requirements of the substrates which were cleaved by the polymerase-independent RNase H activity of E478Q RT are defined.
...
PMID:Comparison of second-strand transfer requirements and RNase H cleavages catalyzed by human immunodeficiency virus type 1 reverse transcriptase (RT) and E478Q RT. 1100 Feb 39


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---