Stereochemical course of catalysis by the Tetrahymena ribozyme

The group I intron from Tetrahymena catalyzes phosphodiester transfer reactions on various RNA substrates. A modified RNA substrate with a phosphorothioate group in one stereoisomeric form at the site of reaction was synthesized in order to determine the stereochemical course of an RNA-catalyzed reaction. The reaction product was digested with a stereospecific nuclease to determine the configuration of the product phosphorothioate. The reaction occurs with inversion of configuration at phosphorus, implying an in-line pathway for the reaction.     

Stereochemistry of RNA cleavage by the Tetrahymena ribozyme and evidence that the chemical step is not rate-limiting

The intervening sequence of the ribosomal RNA precursor of Tetrahymena is a catalytic RNA molecule, or ribozyme. Acting as a sequence-specific endoribonuclease, it cleaves single-stranded RNA substrates with concomitant addition of guanosine. The chemistry of the reaction has now been studied by introduction of a single phosphorothioate in the substrate RNA at the cleavage site. Kinetic studies show no significant effect of this substitution on kcat (rate constant) or Km (Michaelis constant), providing evidence that some step other than the chemical step is rate-limiting. Product analysis reveals that the reaction proceeds with inversion of configuration at phosphorus, consistent with an in-line, SN2 (P) mechanism. Thus, the ribozyme reaction is in the same mechanistic category as the individual displacement reactions catalyzed by protein nucleotidyltransferases, phosphotransferases, and nucleases.     

The intervening sequence RNA of Tetrahymena is an enzyme

A shortened form of the self-splicing ribosomal RNA (rRNA) intervening sequence of Tetrahymena thermophila acts as an enzyme in vitro. The enzyme catalyzes the cleavage and rejoining of oligonucleotide substrates in a sequence-dependent manner with Km = 42 microM and kcat = 2 min-1. The reaction mechanism resembles that of rRNA precursor self-splicing. With pentacytidylic acid as the substrate, successive cleavage and rejoining reactions lead to the synthesis of polycytidylic acid. Thus, the RNA molecule can act as an RNA polymerase, differing from the protein enzyme in that it uses an internal rather than an external template. At pH 9, the same RNA enzyme has activity as a sequence-specific ribonuclease.     

Structural basis of transcription: separation of RNA from DNA by RNA polymerase II

The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network. Formation of the network must occur in the transition from abortive initiation to promoter escape.     

Specific inhibition of nuclear RNA polymerase II by alpha-amanitin

alpha-Amanitin, a toxic substance from the mushroom Amanita phalloides, is a potent inhibitor of DNA-dependent RNA polymerase II (the nucleoplasmic form) from sea urchin, rat liver, and calf thymus. This compound exerts no effect on the activity of polymerase I (nucleolar form) or polymerase III (also nucleoplasmic). The inhibition is due to a specific interaction with polymerase II or with a complex of DNA and polymerase II.     

松材线虫RNA聚合酶基因的RNA干扰研究

克隆了松材线虫(Bursaphelenchus xylophilus)RNA聚合酶基因片段,构建大量表达松材线虫RNA聚合酶基因片段的特异双链RNA(dsRNA)表达载体,并用表达的双链RNA(dsRNA)对松材线虫进行了RNA干扰实验。结果表明,松材线虫浸泡在20℃的RNA聚合酶基因双链RNA(dsRNA)溶液中干扰24 h后再培养12 d,其繁殖倍数为73.2;对照处理的繁殖倍数为322.8,两者的繁殖倍数相差4.4倍;松材线虫浸泡在4℃的RNA聚合酶基因双链RNA(dsRNA)溶液中干扰24 h后再培养12 d,其繁殖倍数为120.8。RNA聚合酶基因的双链RNA(dsRNA)浸泡明显的抑制了松材线虫的繁殖;并且发现利用浸泡法对线虫进行干扰试验,双链RNA(dsRNA)20℃浸泡的干扰效果要好于前人报道4℃浸泡的干扰效果。     

Enzyme from calf thymus degrading the RNA moiety of DNA-RNA Hybrids: effect on DNA-dependent RNA polymerase

An enzyme present in extracts from calf thymus degrades specifically the RNA moiety of DNA-RNA hybrids. Other nucleic acids, such as single- or double-stranded DNA and single- or double-stranded RNA, are not affected to a comparable degree. If prepared free of the hybrid-degrading enzyme, RNA polymerase from calf thymus shows a fivefold increase in activity on denatured DNA as compared to native DNA.     

Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase

High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.