The chemistry of self-splicing RNA and RNA enzymes

Proteins are not the only catalysts of cellular reactions; there is a growing list of RNA molecules that catalyze RNA cleavage and joining reactions. The chemical mechanisms of RNA-catalyzed reactions are discussed with emphasis on the self-splicing ribosomal RNA precursor of Tetrahymena and the enzymatic activities of its intervening sequence RNA. Wherever appropriate, catalysis by RNA is compared to catalysis by protein enzymes.     

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.     

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.     

A labile phosphodiester bond at the ligation junction in a circular intervening sequence RNA

The excised intervening sequence of the Tetrahymena ribosomal RNA precursor mediates its own covalent cyclization in the absence of any protein. The circular molecule undergoes slow reopening at a single phosphodiester bond, the one that was formed during cyclization. The resulting linear molecule has 5'-phosphate and 3'-hydroxyl termini; these are unusual products for RNA hydrolysis but are typical of the other reactions mediated by this molecule. The reopened circle retains cleavage-ligation activity, as evidenced by its ability to undergo another round of cyclization and reopening. The finding that an RNA molecule can be folded so that a specific phosphate can be strained or activated helps to explain how the activation energy is lowered for RNA self-splicing. The proposed mechanisms may be relevant to several other RNA cleavage reactions that are RNA-mediated.     

RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme

A catalytic RNA (ribozyme) derived from an intervening sequence (IVS) RNA of Tetrahymena thermophila will catalyze an RNA polymerization reaction in which pentacytidylic acid (C5) is extended by the successive addition of mononucleotides derived from a guanylyl-(3',5')-nucleotide (GpN). Cytidines or uridines are added to C5 to generate chain lengths of 10 to 11 nucleotides, with longer products being generated at greatly reduced efficiency. The reaction is analogous to that catalyzed by a replicase with C5 acting as the primer, GpNs as the nucleoside triphosphates, and a sequence in the ribozyme providing a template. The demonstration that an RNA enzyme can catalyze net elongation of an RNA primer supports theories of prebiotic RNA self-replication.     

Genetic variation in the human insulin gene

Four recombinant lambda phages containing nucleotide sequences complementary to a cloned human preproinsulin DNA probe have been isolated from human DNA. Restriction analyses in conjunction with Southern hybridizations reveal two types of gene sequences. One isolate of each type was subjected to complete nucleotide sequence determination. The sequences contain the entire preproinsulin messenger RNA region, two intervening sequence. 260 nucleotides upstream from the messenger RNA capping site, and 35 nucleotides beyond the polyadenylate attachment site. Our results strongly suggest that these two gene types are allelic variants of a single insulin gene.     

Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome

The splicing process, which removes intervening sequences from messenger RNA (mRNA) precursors is essential to gene expression in eukaryotic cells. This site-specific process requires precise sequence recognition at the boundaries of an intervening sequence, but the mechanism of this recognition is not understood. The splicing of mRNA precursors occurs in a multicomponent complex termed the spliceosome. Such an assembly of components is likely to play a key role in specifying those sequences to be spliced. In order to analyze spliceosome structure, a stringent approach was developed to obtain splicing complexes free of cellular contaminants. This approach is a form of affinity chromatography based on the high specificity of the biotin-streptavidin interaction. A minimum of three subunits: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles were identified in the 35S spliceosome structure, which also contains the bipartite RNA intermediate of splicing. A 25S presplicing complex contained only the U2 particle. The multiple subunit structure of the spliceosome has implications for the regulation of a splicing event and for its possible catalysis by ribozyme or ribozymes.     

Coupling of Tetrahymena ribosomal RNA splicing to beta-galactosidase expression in Escherichia coli

Splicing of the Tetrahymena ribosomal RNA precursor is mediated by the folded structure of the RNA molecule and therefore occurs in the absence of any protein in vitro. The Tetrahymena intervening sequence (IVS) has been inserted into the gene for the alpha-donor fragment of beta-galactosidase in a recombinant plasmid. Production of functional beta-galactosidase is dependent on RNA splicing in vivo in Escherichia coli. Thus RNA self-splicing can occur at a rate sufficient to support gene expression in a prokaryote, despite the likely presence of ribosomes on the nascent RNA. The beta-galactosidase messenger RNA splicing system provides a useful method for screening for splicing-defective mutations, several of which have been characterized.     

One sequence, two ribozymes: implications for the emergence of new ribozyme folds

We describe a single RNA sequence that can assume either of two ribozyme folds and catalyze the two respective reactions. The two ribozyme folds share no evolutionary history and are completely different, with no base pairs (and probably no hydrogen bonds) in common. Minor variants of this sequence are highly active for one or the other reaction, and can be accessed from prototype ribozymes through a series of neutral mutations. Thus, in the course of evolution, new RNA folds could arise from preexisting folds, without the need to carry inactive intermediate sequences. This raises the possibility that biological RNAs having no structural or functional similarity might share a common ancestry. Furthermore, functional and structural divergence might, in some cases, precede rather than follow gene duplication.     

Variable occurrence of the nrdB intron in the T-even phages suggests intron mobility

The bacteriophage T4 nrdB gene, encoding nucleoside diphosphate reductase subunit B, contains a self-splicing group I intervening sequence. The nrdB intron was shown to be absent from the genomes of the closely related T-even phages T2 and T6. Evidence for variable intron distribution was provided by autocatalytic 32P-guanosine 5'-triphosphate labeling of T-even RNAs, DNA and RNA hybridization analyses, and DNA sequencing studies. The results indicate the nonessential nature of the intron in nrdB expression and phage viability. Furthermore, they suggest that either precise intron loss from T2 and T6 or lateral intron acquisition by T4 occurred since the evolution of these phages from a common ancestor. Intron movement in the course of T-even phage divergence raises provocative questions about the origin of these self-splicing elements in prokaryotes.     

How old is the genetic code? Statistical geometry of tRNA provides an answer

The age of the molecular organization of life as expressed in the genetic code can be estimated from experimental data. Comparative sequence analysis of transfer RNA by the method of statistical geometry in sequence space suggests that about one-third of the present transfer RNA sequence divergence was present at the urkingdom level about the time when archaebacteria separated from eubacteria. It is concluded that the genetic code is not older than, but almost as old as our planet. While this result may not be unexpected, it was not clear until now that interpretable data exist that permit inferences about such early stages of life as the establishment of the genetic code.     

口蹄疫病毒Akesu/58持续感染分离株P1基因的克隆及序列分析

以持续感染模型动物牦牛的食道/咽部分离物(O/P液)为材料,采用RT-PCR法,扩增和克隆了P1结构蛋白基因的全序列。序列测定和分析结果表明,持续感染分离株的序列与Akesu/58参考毒株的序列相比,其同源性为86.5 %,而与china/99的同源性高达89.4 %。氨基酸差异分析表明,氨基酸相应的密码子中T-C或A-G互变频率较高,这可能是导致氨基酸改变的主要因素。     

A specific amino acid binding site composed of RNA

A specific, reversible binding site for a free amino acid is detectable on the intron of the Tetrahymena self-splicing ribosomal precursor RNA. The site selects arginine among the natural amino acids, and prefers the L- to the D-amino acid. The dissociation constant is in the millimolar range, and amino acid binding is at or in the catalytic rG splicing substrate site. Occupation of the G site by L-arginine therefore inhibits splicing by inhibiting the binding of rG, without inhibition of later reactions in the splicing reaction sequence. Arginine binding specificity seems to be directed at the side chain and the guanidino radical, and the alpha-amino and carboxyl groups are dispensable for binding. The arginine site can be placed within the G site by structural homology, with consequent implications for RNA-amino acid interaction, for the origin of the genetic code, for control of RNA activities, and for further catalytic capabilities for RNA.     

A subset of yeast snRNA's contains functional binding sites for the highly conserved Sm antigen

Autoimmune sera of the Sm specificity react with the major class of small nuclear RNA (snRNA)-containing ribonucleoprotein particles (snRNP's) from organisms as evolutionarily divergent as insects and dinoflagellates but have been reported not to recognize snRNP's from yeast. The Sm antigen is thought to bind to a conserved snRNA motif that includes the sequence A(U3-6)G. The hypothesis was tested that yeast also contains functional analogues of Sm snRNA's, but that the Sm binding site in the RNA is more strictly conserved than the Sm antigenic determinant. After microinjection of labeled yeast snRNA's into Xenopus eggs or oocytes, two snRNA's from Saccharomyces cerevisiae become strongly immunoprecipitable with human auto-antibodies known as anti-Sm. These each contain the sequence A(U5-6)G, are essential for viability, and are constituents of the spliceosome. At least six other yeast snRNA's do not become immunoprecipitable and lack this sequence; these non-Sm snRNA's are all dispensable.