Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1-871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1-RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1-RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1-RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding.
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PMID:The p80 homology region of TEP1 is sufficient for its association with the telomerase and vault RNAs, and the vault particle. 1570 61

Pausing by reverse transcriptase (RT) during retroviral replication increases the frequency of homologous strand transfer, nucleotide misincorporation, and non-templated nucleotide addition. Pausing frequency increases at sites of DNA damage or upon incorporation of nucleotide analogs with steric barriers. These lesions thus likely stimulate mutations leading to resistant viral strains that escape drug treatments or immune surveillance. To study the response of retroviral RTs to bulky 2' adducts, a ribozyme-catalyzed reaction was used to generate an RNA template strand containing a thiophosphate adduct at a specific 2'-hydroxyl located upstream from a polyadenosine sequence. Subsequent alkylation increased the size of the adduct. Polymerization readthrough efficiencies were compared for mature RTs derived from HIV-1 (p66/p51), AMV (p95/p63), MMLV (p80 monomer), and a truncated version of HIV-1 RT lacking the RNase H domain (p51/p51 homodimer). Readthrough at the 2' lesion was markedly greater for the p51/p51 homodimer of HIV-1 RT than for the other enzymes, suggesting that the presence of the RNase H domain increases the probability that the modified primer/template will encounter a barrier to translocation. Comparison to published structures suggests potential unfavorable interactions between the 2' adduct and W24, F61, I63, D76, and R78 in the fingers domain of the RT. We propose that the enhanced readthrough observed upon RNase H domain deletion alters the trajectory of the primer/template in this region that diminishes steric and electrostatic clash with these residues. The template also included a penta-adenosine sequence that induced pausing in the order MMLV > HIV-1 (p66/p51) > AMV ~ HIV-1 (p51/p51).
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PMID:HIV-1 reverse transcriptase pausing at bulky 2' adducts is relieved by deletion of the RNase H domain. 1739 57