The computer simulation of RNA folding involving pseudoknot formation.

The algorithm and the program for the prediction of RNA secondary structure with pseudoknot formation have been proposed. The algorithm simulates stepwise folding by generating random structures using Monte Carlo method, followed by the selection of helices to final structure on the basis of both their probabilities of occurrence in a random structure and free energy parameters. The program versions have been tested on ribosomal RNA structures and on RNAs with pseudoknots evidenced by experimental data. It is shown that the simulation of folding during RNA synthesis improves the results. The introduction of pseudoknot formation permits to predict the pseudoknotted structures and to improve the prediction of long-range interactions. The computer program is rather fast and allows to predict the structures for long RNAs without using large memory volumes in usual personal computer.     

Rearrangement of structured RNA via branch migration structures catalysed by the highly related DEAD-box proteins p68 and p72

RNA helicases, like their DNA-specific counterparts, can function as processive enzymes, unwinding RNA with a defined step size in a unidirectional fashion. Recombinant nuclear DEAD-box protein p68 and its close relative p72 are reported here to function in a similar fashion, though the processivity of both RNA helicases appears to be limited to only a few consecutive catalytic steps. The two proteins resemble each other also with regard to other biochemical properties. We have found that both proteins exhibit an RNA annealing in addition to their helicase activity. By using both these activities the enzymes are able in vitro to catalyse rearrangements of RNA secondary structures that otherwise are too stable to be resolved by their low processive helicase activities. RNA rearrangement proceeds via protein induced formation and subsequent resolution of RNA branch migration structures, whereby the latter step is dependent on ATP hydrolysis. The analysed DEAD-box proteins are reminiscent of certain DNA helicases, for example those found in bacteriophages T4 and T7, that catalyse homologous DNA strand exchange in cooperation with the annealing activity of specific single strand binding proteins.     

Mass spectrometry reveals stable modules in holo and apo RNA polymerases I and III

RNA polymerases are essential enzymes which transcribe DNA into RNA. Here, we obtain mass spectra of the cellular forms of apo and holo eukaryotic RNA polymerase I and III, defining their composition under different solution conditions. By recombinant expression of subunits within the initiation heterotrimer of Pol III, we derive an interaction network and couple this data with ion mobility data to define topological restraints. Our data agree with available structural information and homology modeling and are generally consistent with yeast two hybrid data. Unexpectedly, elongation complexes of both Pol I?and III destabilize the assemblies compared with their apo counterparts. Increasing the pH and ionic strength of apo and holo forms of Pol I and Pol III leads to formation of at least ten stable subcomplexes for both enzymes. Uniquely for Pol III many subcomplexes contain only one of the two largest catalytic subunits. We speculate that these stable subcomplexes represent putative intermediates in assembly pathways.     

The catalytic diversity of RNAs

The natural RNA enzymes catalyse phosphate-group transfer and peptide-bond formation. Initially, metal ions were proposed to supply the chemical versatility that nucleotides lack. In the ensuing decades, structural and mechanistic studies have substantially altered this initial viewpoint. Whereas self-splicing ribozymes clearly rely on essential metal-ion cofactors, self-cleaving ribozymes seem to use nucleotide bases for their catalytic chemistry. Despite the overall differences in chemical features, both RNA and protein enzymes use similar catalytic strategies.     

Uncoupling of Protein and Ribonucleic Acid Synthesis by 5′,5′,5′-Trifluoroleucine in Salmonella typhimurium

The addition of 5',5',5'-trifluoroleucine (fluoroleucine) to leucine auxotrophs of Salmonella typhimurium permitted protein but not ribonucleic acid (RNA) synthesis to continue after leucine depletion. The uncoupling of the formation of these macromolecules by fluoroleucine was apparent if RNA and protein synthesis was measured either by the uptake of radioactive precursors or by direct chemical determinations. The analogue did not appear to be an inhibitor of RNA formation, since it was as effective as leucine in permitting RNA synthesis in a leucine auxotroph upon the addition of small amounts of chloramphenicol. In contrast to these data, fluoroleucine allowed continued protein and RNA formation in a leucine auxotroph of Escherichia coli strain W. In addition, contrary to the results obtained with S. typhimurium, the analogue replaced leucine for repression of the leucine bio-synthetic enzymes as well as the isoleucine-valine enzymes. We propose that these ambivalent effects of fluoroleucine on repression and RNA and protein synthesis in the two strains are due to differences in the ability of the analogue to attach to the various species of leucine transfer RNA.     

Understanding the transition states of phosphodiester bond cleavage: insights from heavy atom isotope effects

The nucleotides of DNA and RNA are joined by phosphodiester linkages whose synthesis and hydrolysis are catalyzed by numerous essential enzymes. Two prominent mechanisms have been proposed for RNA and protein enzyme catalyzed cleavage of phosphodiester bonds in RNA: (a) intramolecular nucleophilic attack by the 2'-hydroxyl group adjacent to the reactive phosphate; and (b) intermolecular nucleophilic attack by hydroxide, or other oxyanion. The general features of these two mechanisms have been established by physical organic chemical analyses; however, a more detailed understanding of the transition states of these reactions is emerging from recent kinetic isotope effect (KIE) studies. The recent data show interesting differences between the chemical mechanisms and transition state structures of the inter- and intramolecular reactions, as well as provide information on the impact of metal ion, acid, and base catalysis on these mechanisms. Importantly, recent nonenzymatic model studies show that interactions with divalent metal ions, an important feature of many phosphodiesterase active sites, can influence both the mechanism and transition state structure of nonenzymatic phosphodiester cleavage. Such detailed investigations are important because they mimic catalytic strategies employed by both RNA and protein phosphodiesterases, and so set the stage for explorations of enzyme-catalyzed transition states. Application of KIE analyses for this class of enzymes is just beginning, and several important technical challenges remain to be overcome. Nonetheless, such studies hold great promise since they will provide novel insights into the role of metal ions and other active site interactions.     

RNA解旋酶和应激颗粒

应激颗粒（stress granules, SGs）是细胞在环境压力刺激下停止蛋白质翻译后,mRNA与多种细胞蛋白组装而成的胞质颗粒结构.RNA 解旋酶家族作为生物体内普遍存在的一类高度保守的蛋白质酶类,参与了RNA代谢各个环节,近年来其家族成员被陆续发现是一类新的SG重要组分.本文综述了RNA解旋酶参与应激颗粒形成过程,RNA解旋酶家族蛋白的结构和其参与应激颗粒形成的研究进展.     

A possible origin of RNA catalysis in multienzyme complexes

Numerous attempts have recently been made to ascribe a preeminent role to RNA enzymes in primitive life systems. A model is proposed in which coenzyme-dependent RNA enzymes were initially organized in multienzyme complexes featuring (1) the continuous attachment of substrates to CoA-like carriers, as in fatty acid synthesis; and (2) the ordering of RNA enzymes via mRNA-like instructional strands. In this format, RNA enzymes would not have been required to recognized and specifically bind soluble substrates. The enzymes in this case may have required far less complexity than contemporary protein enzymes and thus less genetic information for their synthesis. An analogy is made between the proposed scheme and the protein translation mechanism, for which it may have been an evolutionary precursor.     

Yeast Rrp8p,a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA

Ribosomal RNA undergoes various modifications to optimize ribosomal structure and expand the topological potential of RNA. The most common nucleotide modifications in ribosomal RNA (rRNA) are pseudouridylations and 2′-O methylations (Nm), performed by H/ACA box snoRNAs and C/D box snoRNAs, respectively. Furthermore, rRNAs of both ribosomal subunits also contain various base modifications, which are catalysed by specific enzymes. These modifications cluster in highly conserved areas of the ribosome. Although most enzymes catalysing 18S rRNA base modifications have been identified, little is known about the 25S rRNA base modifications. The m¹A modification at position 645 in Helix 25.1 is highly conserved in eukaryotes. Helix formation in this region of the 25S rRNA might be a prerequisite for a correct topological framework for 5.8S rRNA to interact with 25S rRNA. Surprisingly, we have identified ribosomal RNA processing protein 8 (Rrp8), a nucleolar Rossman-fold like methyltransferase, to carry out the m¹A base modification at position 645, although Rrp8 was previously shown to be involved in A2 cleavage and 40S biogenesis. In addition, we were able to identify specific point mutations in Rrp8, which show that a reduced S-adenosyl-methionine binding influences the quality of the 60S subunit. This highlights the dual functionality of Rrp8 in the biogenesis of both subunits.     

In vitro reconstitution reveals a key role of human mitochondrial EXOG in RNA primer processing

The removal of RNA primers is essential for mitochondrial DNA (mtDNA) replication. Several nucleases have been implicated in RNA primer removal in human mitochondria, however, no conclusive mechanism has been elucidated. Here, we reconstituted minimal in vitro system capable of processing RNA primers into ligatable DNA ends. We show that human 5′-3′ exonuclease, EXOG, plays a fundamental role in removal of the RNA primer. EXOG cleaves short and long RNA-containing flaps but also in cooperation with RNase H1, processes non-flap RNA-containing intermediates. Our data indicate that the enzymatic activity of both enzymes is necessary to process non-flap RNA-containing intermediates and that regardless of the pathway, EXOG-mediated RNA cleavage is necessary prior to ligation by DNA Ligase III. We also show that upregulation of EXOG levels in mitochondria increases ligation efficiency of RNA-containing substrates and discover physical interactions, both in vitro and in cellulo, between RNase H1 and EXOG, Pol γA, Pol γB and Lig III but not FEN1, which we demonstrate to be absent from mitochondria of human lung epithelial cells. Together, using human mtDNA replication enzymes, we reconstitute for the first time RNA primer removal reaction and propose a novel model for RNA primer processing in human mitochondria.     

The Saccharomyces cerevisiae Pus2 protein encoded by YGL063w ORF is a mitochondrial tRNA:Psi27/28-synthase

The RNA:pseudouridine (Psi)-synthase family is one of the most complex families of RNA modification enzymes. Ten genes encoding putative RNA:Psi-synthases have been identified in S. cerevisiae. Most of the encoded enzymes have been characterized experimentally. Only the putative RNA:Psi-synthase Pus2p (encoded by the YGL063w ORF) had no identified substrate. Here, we analyzed Psi residues in cytoplasmic and mitochondrial tRNAs extracted from S. cerevisiae strains, carrying disruptions in the PUS1 and/or PUS2 ORFs. Our results demonstrate that Pus2p is a mitochondrial-specific tRNA:Psi-synthase acting at positions 27 and 28 in tRNAs. The importance of the Asp56 residue in the conserved ARTD motif of the Pus2p catalytic site is demonstrated in vivo. Interestingly, in spite of the absence of a characteristic N-terminal targeting signal, our data strongly suggest an efficient and rapid targeting of Pus2p in yeast mitochondria. In contradiction with the commonly held idea that a unique nuclear gene encodes the enzyme required for both cytoplasmic and mitochondrial tRNA modifications, here we show the existence of an enzyme specifically dedicated to mitochondrial tRNA modification (Pus2p), the corresponding modification in cytoplasmic tRNAs being catalyzed by another protein (Pus1p).     

Polyamine synthesis during lymphocyte activation : Induction of ornithine decarboxylase and S-adenosyl methionine decarboxylase

Lymphocyte stimulation by phytohaemagglutinin (PHA) is accompanied by marked increases in the activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase, two key enzymes for the synthesis of polyamines. Both enzymes increase in a biphasic manner, with the rises in S-adenosyl methionine decarboxylase preceding the increases in ornithine decarboxylase. The initial rises precede the initiation of DNA synthesis, and seem to correlate with the increased rate of ribosomal RNA synthesis. Selective inhibition of ribosomal RNA synthesis inhibits the increases in the activity of both enzymes, especially ornithine decarboxylase, more than the increase in the overall rate of protein synthesis.Both enzymes are metabolically unstable and have half-lives of less than 1 h, although the half-life of ornithine decarboxylase depends on the amino acid concentration in the culture medium. While effects of PHA on the stability of the enzymes have not been ruled out, at least part of the PHA-dependent increases in activity are due to increased synthesis or activation of the enzymes. The synthesis of S-adenosyl-methionine decarboxylase declines rapidly after inhibition of RNA synthesis, but ornithine decarboxylase activity declines at about the same rate as protein synthesis as a whole.The activities of both enzymes also increase during lymphocyte stimulation by concanavalin A, lentil extract and staphylococcal filtrate.     

Purification and subunit structure of RNA polymerases I and II from Dictyostelium discoideum vegetative cells

Summary A purification procedure to obtain RNA polymerases I (or A) and II (or B) from Dictyostelium discoideum amoeba has been developed. The enzymes were solubilized from purified nuclei and separated by DEAF-Sephadex chromatography. RNA polymerases I and II were further purified by a second chromatography on DEAE-Sephadex followed by chromatographies on phosphocellulose and heparin-sepharose. The specific activities of purified RNA polymerases I and II are 92 units/ mg protein and 70 units/ mg protein, respectively. The subunit structure of both RNA polymerases were analyzed by polyacrylamide gel electrophoresis under denaturing conditions after glycerol gradient centrifugation of the enzymes. The putative subunits of RNA polymerase I have molecular weights of 180 000,125 000,43 000,40 000,34 000, 31 000, 25 000,19 000, 17 000 and 14 000. The putative subunits of RNA polymerase II have molecular weights of 200 000 (170 000), 130 000, 33 000, 25 000, 19 000, 17 000, 15 000, 13 000. There are three polypeptides with common molecular weight in Dictyostelium RNA polymerases I and 11. The subunit of 25 000 daltons of both enzymes has common immunological determinants with RNA polymerase II from crustacean Artemia.Abbreviations TLCK tosyl-lysine-chloromethyl-ketone - DPT diazophenylthioether