In two recent articles by Nitta et al. (1998a, 1998b), evidence was presented that in vitro-transcribed protein-free 23S rRNA of Escherichia coli (and its individual domains) can catalyze peptide bond formation. The implications of these studies are considerable because demonstration of catalytic activity associated with protein-free 23S rRNA would be fundamental to our understanding of the mechanism and evolution of protein synthesis (Noller et al., 1992; Khaitovich et al., 1999).

A set of free energy values is suggested for RNA H-pseudoknot loops. The parameters are adjusted to be consistent with the theory of polymer thermodynamics and known data on pseudoknots. The values can be used for estimates of pseudoknot stabilities and computer predictions of RNA structures.

In vitro transcription with T7 RNA polymerase is one of the most powerful tools in RNA research. This is mainly linked to the easy preparation of the enzyme, the quite unlimited range of sizes and sequences of the RNA that can be synthesized, as well as the efficiency and accuracy of synthesis. So far only two major limitations are common knowledge, namely poor transcription efficiency of non-G-rich initial sequences (Dunn & Studier, 1983) and 3′-end heterogeneities of the transcripts by 1 or 2 nt (Milligan et al., 1987; Draper et al., 1988; Kholod et al., 1998). These drawbacks have been overcome in most cases by improving the initiating sequence and/or purifying the transcription products to single-nucleotide resolution when necessary. The Uhlenbeck laboratory has published the first evidence for shifty errors by the T7 RNA polymerase at the very 5′ end of the synthesized RNA, an as yet unsuspected event (Pleiss et al., 1998). They demonstrated that in the particular case of tRNA sequences starting with G-rich stretches, the enzyme incorporates additional nontemplated G residues in up to 30% of the transcripts. The authors point to the fact that these errors, in combination with the 3′-end heterogeneity common to T7 transcripts, may be crucial, for instance, for functional studies of tRNAs, because some in vitro-transcribed tRNA molecules of rigorously correct final size may be erroneously considered as functional.

Analysis of the sequence of the mitochondrial cyt b gene of Teratocephalus lirellus revealed multiple frame shift events. As these sites coincided with homopolymeric tracks, sequencing was repeated and chromatograms were screened for poorly resolved peaks. No evidence for sequencing errors was found. We therefore reverse transcribed mRNA, using M-MLV reverse transcriptase and two primers for strongly conserved regions located anteriorly (CbF1: >5′-GTTTTGGAATGTTGGAAGGCTTTTAGG-3′) and posteriorly (CbR1: <5′-CCCAGATATCACCATAGCCAAATAGACG-3′) in the gene. The cDNA product was PCR amplified and sequenced, and compared with the gDNA sequences. This revealed insertions of a single U in eight sites in the mRNA sequence (Fig. 1).

Translation of UGA as selenocysteine requires specific RNA secondary structures in the mRNAs of selenoproteins. These elements differ in sequence, structure, and location in the mRNA, that is, coding versus 3′ untranslated region, in prokaryotes, eukaryotes, and archaea. Analyses of eukaryotic selenocysteine insertion sequence (SECIS) elements via computer folding programs, mutagenesis studies, and chemical and enzymatic probing has led to the derivation of a predicted consensus structural model for these elements. This model consists of a stem-loop or hairpin, with conserved nucleotides in the loop and in a non-Watson–Crick motif at the base of the stem. However, the sequences of a number of SECIS elements predict that they would diverge from the consensus structure in the loop region. Using site-directed mutagenesis to introduce mutations predicted to either disrupt or restore structure, or to manipulate loop size or stem length, we show that eukaryotic SECIS elements fall into two distinct classes, termed forms 1 and 2. Form 2 elements have additional secondary structures not present in form 1 elements. By either insertion or deletion of the sequences and structures distinguishing the two classes of elements while maintaining appropriate loop size, conversion of a form 1 element to a functional form 2-like element and of a form 2 to a functional form 1-like element was achieved. These results suggest commonality of function of the two classes. The information obtained regarding the existence of two classes of SECIS elements and the tolerances for manipulations of stem length and loop size should facilitate designing RNA molecules for obtaining high-resolution structural information about these elements.

Conserved octanucleotide sequences located upstream of two major potato virus X (PVX) subgenomic RNAs (sgRNAs), as well as elements in the 5′ end of the genome, affect accumulation of sgRNA. To determine if complementarity between these sequences is important for PVX RNA accumulation, we analyzed the effects of mutations within these elements and compensatory mutations in a tobacco protoplast system and in plants. Mutations in the 5′ nontranslated region (NTR mutants) that reduced complementarity resulted in lower genomic RNA (gRNA) and sgRNA levels, whereas mutations to the octanucleotide elements affected only the corresponding sgRNA levels. However, for both the NTR and octanucleotide mutants, the extent of reductions in RNA levels did not directly correlate with the degree of complementarity, suggesting that the sequences of these elements are also important. Mutants containing changes in the NTR and compensatory changes in one of the octanucleotide elements restored levels of gRNA and the other sgRNA species with an unaltered octanucleotide element to those of wild-type. Although compensatory changes significantly increased levels of the sgRNA species with the modified octanucleotide element, levels were not restored to those of wild-type. Our data indicate that long distance RNA–RNA interactions and the sequences of the interacting elements are required for PVX plus-strand RNA accumulation.

Folding pathways of large RNAs are poorly understood. We have addressed this question by hybridizing in vitro transcripts, which varied in size, to an array of antisense oligonucleotides. All transcripts included a common sequence and all but one shared the same start-point; the other had a small deletion of the 5′ end. Minimal free energy calculations predicted quite different folds for these transcripts. However, hybridization to the array showed predominant features that were shared by transcripts of all lengths, though some oligonucleotides that hybridized strongly to the short transcripts gave weak interaction with longer transcripts. A full-length RNA fragment that had been denatured by heating and allowed to cool slowly gave the same hybridization result as a shorter transcript. Taken together, these results support theories that RNA folding creates local stable states that are trapped early in the transcription or folding process. As the transcript elongates, interactions are added between regions that are transcribed early and those transcribed late. The method here described helps in identifying regions in the transcripts that take part in long-range interactions.

Uridine (U) insertion/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa is a posttranscriptional process mediated by guide RNAs (gRNAs). The gRNAs direct the precise insertion and deletion of Us by a cleavage–ligation mechanism involving base pairing. We show that a cognate gRNA in cis at the 3′ end of a preedited NADH dehydrogenase 7 (ND7) mRNA substrate can direct U insertions at editing site 1 when incubated with a mitochondrial lysate from Leishmania tarentolae. The efficiency of gRNA-dependent U insertion mediated by a cis-acting gRNA is greater on a molar basis than that for a trans-acting gRNA, as expected for a unimolecular gRNA:mRNA interaction. Blocking the 3′ end of a cis-acting gRNA lacking a 3′ oligo[U] tail has no effect on gRNA-dependent U insertions, nor does providing the gRNA in cis upstream of the mRNA, confirming the previous observation that the terminal 2′- and 3′-hydroxyls of the gRNA are not involved in U insertion activity. These results also establish that the oligo[U] tail is not required for U insertion in vitro. Increasing the extent of base pairing between the 3′ end of the gRNA and the 5′ end of the mRNA significantly increases in vitro gRNA-dependent U insertion at site 1, presumably by maintaining the mRNA 5′ cleavage fragment within the editing complex. We speculate that, in vivo, protein:RNA and/or protein:protein interactions may be responsible for maintaining the mRNA 5′ cleavage fragment in close proximity to the mRNA 3′ cleavage fragment, and that such interactions may be rate limiting in vitro.

The acceptor stem of Escherichia coli tRNAAla, rGGGGCUA[bull ]rUAGCUCC (ALAwt), contains the main identity element for the correct aminoacylation by the alanyl tRNA synthetase. The presence of a G3[bull ]U70 wobble base pair is essential for the specificity of this reaction, but there is a debate whether direct minor-groove contact with the 2-amino group of G3 or a distortion of the acceptor stem induced by the wobble pair is the critical feature recognized by the synthetase. We here report the structure analysis of ALAwt at near-atomic resolution using twinned crystals. The crystal lattice is stabilized by a novel strontium binding motif between two cis-diolic O3′-terminal riboses. The two independent molecules in the asymmetric unit of the crystal show overall A-RNA geometry. A comparison with the crystal structure of the G3-C70 mutant of the acceptor stem (ALAC70) determined at 1.4 Å exhibits a modulation in ALAwt of helical twist and slide due to the wobble base pair, but no recognizable distortion of the helix fragment distant from the wobble base pair. We suggest that a highly conserved hydration pattern in both grooves around the G3[bull ]U70 wobble base pair may be functionally significant.

In yeast, the 5′ end of the mature 18S rRNA is generated by endonucleolytic cleavage at site A1, the position of which is specified by two distinct signals. An evolutionarily conserved sequence immediately upstream of the cleavage site has previously been shown to constitute one of these signals. We report here that a conserved stem-loop structure within the 5′ region of the 18S rRNA is recognized as a second positioning signal. Mutations predicted to either extend or destabilize the stem inhibited the normal positioning of site A1 from within the 18S rRNA sequence, as did substitution of the loop nucleotides. In addition, these mutations destabilized the mature 18S rRNA, indicating that recognition of the stem-loop structure is also required for 18S rRNA stability. Several mutations tested reduced the efficiency of pre-rRNA cleavage at site A1. There was, however, a poor correlation between the effects of the different mutations on the efficiency of cleavage and on the choice of cleavage site, indicating that these involve recognition of the stem-loop region by distinct factors. In contrast, the cleavages at sites A1 and A2 are coupled and the positioning signals appear to be similar, suggesting that both cleavages may be carried out by the same endonuclease.

The mouse c-src gene contains a short neuron-specific exon, N1. N1 exon splicing is partly controlled by an intronic splicing enhancer sequence that activates splicing of a heterologous reporter exon in both neural and nonneural cells. Here we attempt to dissect all of the regulatory elements controlling the N1 exon and examine how these multiple elements work in combination. We show that the 3′ splice site sequence upstream of exon N1 represses the activation of splicing by the downstream intronic enhancer. This repression is stronger in nonneural cells and these two regulatory sequences combine to make a reporter exon highly cell-type specific. Substitution of the 3′ splice site of this test exon with sites from other exons indicates that activation by the enhancer is very dependent on the nature of the upstream 3′ splice site. In addition, we identify a previously uncharacterized purine-rich sequence within exon N1 that cooperates with the downstream intronic enhancer to increase exon inclusion. Finally, different regulatory elements were tested in multiple cell lines of both neuronal and nonneuronal origin. The individual splicing regulatory sequences from the src gene vary widely in their activity between different cell lines. These results demonstrate how a simple cassette exon is controlled by a variety of regulatory elements that only in combination will produce the correct tissue specificity of splicing.

This is in reference to the paper by Nitta et al. published in RNA in March, 1998.