The current nomenclature system of group I introns (see Cech, 1988; Michel & Westhof, 1990) has become insufficient to distinguish and categorize the complex collection of more than 900 group I introns in ribosomal DNA (rDNA) of nuclear, mitochondrial, chloroplast, and eubacterial genomes (http://www.rna.icmb.utexas.edu/; GenBank; our unpubl. results) in a rational way. The majority of these group I introns (∼750) are found in nuclear rDNA of fungi and protists, but the distribution appears highly scattered since most species analyzed lack introns. Many of the rDNA introns are optional among strains of a particular species or between closely related species, and some have been shown in experimental settings to be true mobile genetic elements (see Belfort & Roberts, 1997). All group I rDNA introns are found at a limited number of insertion sites (∼75) in highly conserved regions of the small subunit (SSU) and large subunit (LSU) rRNA genes, and some of these sites (∼10) are shared by introns from the nuclei, mitochondria, or chloroplasts. There are numerous examples of multiple group I introns in a single rRNA gene, and as many as eight nuclear introns have been noted in the SSU rDNA of the lichen ascomycete Lecanora dispersa (accession number L37734) and in the LSU rDNA of the myxomycete Fuligo septica (our unpubl. results). Finally, group I introns that occupy the same site in rDNA, but in distantly related hosts, tend to share a number of structural features as well as high levels of primary sequence similarities compared to introns at different insertion sites (e.g., Suh et al., 1999).

A yeast three-hybrid system was employed to analyze interactions in vivo between H1 RNA, the RNA subunit of human nuclear RNase P, and eight of the protein subunits of the enzyme. The genetic analysis indicates that subunits Rpp21, Rpp29, Rpp30, and Rpp38 interact directly with H1 RNA. The results of direct UV crosslinking studies of the purified RNase P holoenzyme confirm the results of the three-hybrid assay.

Although the decoding rules have been largely elucidated, the physical-chemical reasons for the “correctness” of codon:anticodon duplexes have never been clear. In this work, on the basis of the available data, we propose that the correct codon:anticodon duplexes are those whose formation and interaction with the ribosomal decoding center are not accompanied by uncompensated losses of hydrogen and ionic bonds. Other factors such as proofreading, base–base stacking and aminoacyl–tRNA concentration contribute to the efficiency and accuracy of aminoacyl–tRNA selection, and certainly these factors are important; but we suggest that analyses of hydrogen and ionic bonding alone provides a robust first-order approximation of decoding accuracy. Thus our model can simplify predictions about decoding accuracy and error. The model can be refined with data, but is already powerful enough to explain all of the available data on decoding accuracy. Here we predict which duplexes should be considered correct, which duplexes are responsible for virtually all misreading, and we suggest an evolutionary scheme that gave rise to the mixed boxes of the genetic code.

We have detected by nucleotide analog interference mapping (NAIM) purine N7 functional groups in Escherichia coli RNase P RNA that are important for tRNA binding under moderate salt conditions (0.1 M Mg2+, 0.1 M NH4+). The majority of identified positions represent highly or universally conserved nucleotides. Our assay system allowed us, for the first time, to identify c7-deaza interference effects at two G residues (G292, G306). Several c7-deazaadenine interference effects (A62, A65, A136, A249, A334, A351) have also been identified in other studies performed at very different salt concentrations, either selecting for substrate binding in the presence of 0.025 M Ca2+ and 1 M NH4+ or self-cleavage of a ptRNA–RNase P RNA conjugate in the presence of 3 M NH4+ or Na+. This indicates that these N7 functional groups play a key role in the structural organization of ribozyme–substrate and –product complexes. We further observed that a c7-deaza modification at A76 of tRNA interferes with tRNA binding to and ptRNA processing by E. coli RNase P RNA. This finding combined with the strong c7-deaza interference at G292 of RNase P RNA supports a model in which substrate and product binding to E. coli RNase P RNA involves the formation of intermolecular base triples (A258-G292-C75 and G291-G259-A76).

IF3 is essential for ensuring the fidelity of the initiation step of translation in bacterial cells. Mutations at residues R99 and R131 in the C-terminal domain of the factor have previously been shown to increase initiation from the noncanonical GUA codon. Here we show that these mutant forms of IF3 fail to discriminate against initiation from many different non-AUG codons. They also enhance the activity of mutant tRNAs carrying changes in the three consecutive G-C pairs that are conserved in the anticodon stem of initiator tRNAs. In addition, the IF3 mutants stimulate initiations from leaderless mRNAs and from internal initiation codons, in the absence of any SD–anti-SD interaction. These results indicate that IF3 ensures the accuracy of initiation by inspecting both the codon–anticodon pairing and unique features of the initiator tRNA as well as suppressing initiation from other potential start sites within the mRNA.

The packaging signal present in influenza viral RNA molecules is shown not to constitute a separate structural element, but to reside within the 5′-bulged promoter structure, as caused by the central unpaired residue A10 in its 5′ branch. Upon insertion of two uridine residues in the 3′ branch opposite A10, the minus-strand viral RNA (vRNA) promoter is converted into a 3′-bulged structure, whereas the plus-strand cRNA promoter instead adopts the 5′-bulged conformation. In this promoter variant it is exclusively the cRNA that is found packaged in the progeny virions. Upon insertion of only a single uridine nucleotide opposite 5′A10, the two debulged structures of the vRNA and cRNA promoters are rendered identical, and both vRNA and cRNA molecules are packaged indiscriminately, in a 1:1 ratio, but at lower rates. We propose that the binding interactions of viral polymerase with either of the two differently bulged vRNA and cRNA promoter structures result in two different conformations of the enzyme protein. Only the 5′ bulged RNA-associated polymerase conformation appears to be recognized for nuclear export, which depends on nuclear matrix protein M1 and nonstructural protein NS2. And the respective wild-type vRNP- or insertion mutant cRNP complex is observed to enter the cytoplasm and hence is included in the viral encapsidation process, which takes place at the plasma membrane.

This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917, blocks pseudouridine formation and inhibits growth. We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated. In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive. In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these pseudouridines. Pseudoreversion, which has previously been shown to restore growth without pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.

tmRNA facilitates a novel translation, trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA's tag sequence. The mechanism underlying resumption of translation at a definite position is not known. In the present study, the effects of mutations around the initiation point of the tag-encoding sequence of Escherichia coli tmRNA on the efficiency and the frame of tag translation were assessed by measuring the incorporations of several amino acids into in vitro poly (U)-dependent tag-peptide synthesis. One-nucleotide insertions within the tag-encoding region did not shift the frame of tag translation. Any 1-nt deletion within the span of −5 to −1, but not at −6, made the frame of tag translation heterologous. Positions at which a single base substitution caused a decrease of trans-translation efficiency were concentrated within the span of −4 to −2. In particular, an A-4 to C-4 mutation seriously damaged the trans-translation, although this mutant retained normal aminoacylation and ribosome-binding abilities. A possible stem and loop structure around this region was not required for trans-translation. It was concluded that the tag translation requires the primary sequence encompassing −6 to +1, in which the central 3 nt, A-4, G-3, and U-2, play an essential role. It was also found that several base substitutions within the span of −6 to −1 extensively shifted the tag-initiation point by −1.

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5′ exon before the first step of splicing and with the 5′ and 3′ exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5′ and 3′ exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5′ exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5′ exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5′ splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5′ exon in two steps during its targeting to the 5′ splice site.

Using a series of point mutations in chimeric reporter gene constructs consisting of the 5′ regions of the Chlamydomonas chloroplast rbcL or atpB genes fused 5′ to the coding sequence of the bacterial uidA (GUS) gene, RNA-stabilizing sequence elements were identified in vivo in the 5′ untranslated regions (5′ UTRs) of transcripts of the chloroplast genes rbcL and atpB in Chlamydomonas reinhardtii. In chimeric rbcL 5′ UTR:GUS transcripts, replacement of single nucleotides in the 10-nt sequence 5′-AUUUCCGGAC-3′, extending from positions +38 to +47 relative to the transcripts' 5′ terminus, shortened transcript longevity and led to a reduction in transcript abundance of more than 95%. A similar mutational analysis of atpB 5′ UTR:GUS transcripts showed that the 12-nt atpB 5′ UTR sequence 5′-AUAAGCGUUAGU-3′, extending from position +31 to position +42, is important for transcript stability and transcript accumulation in the chloroplast of Chlamydomonas. We discuss how the 5′ UTR sequence elements, which are predicted to be part of RNA secondary structures, might function in RNA stabilization.

The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to limit poly(A) tail length on pre-mRNA to <20 nt. Functional PLEs are present in a number of genes, underscoring the generality of this control mechanism. The current study sought to define further the position requirements for poly(A) length regulation and the core sequence that comprises a PLE. Increasing the spacing between the PLE and the upstream 3′ splice site or between the PLE and the downstream AAUAAA had no effect on poly(A) length control. However, moving the PLE from the terminal exon to either an upstream exon or intron eliminated poly(A) length control. Poly(A) length control was further evaluated using a battery of constructs in which the PLE was maintained in the terminal exon, but where upstream introns were either deleted, modified, or replaced with a polypyrimidine tract. Poly(A) length control was retained in all cases, indicating that the key feature is the presence of the PLE in the terminal exon. A battery of mutations demonstrated the importance of the 5′ pyrimidine-rich portion of the element. Finally, UV crosslinking experiments identified an ∼62-kDa protein in Hela nuclear extract that binds to a wild-type 23-nt PLE RNA oligonucleotides but not to a mutated nonfunctional form of the element.

We previously reported a bifunctional ribozyme that catalyzes self-aminoacylation and subsequent acyl-transfer to a tRNA. The ribozyme selectively recognizes a biotinyl-glutamine substrate, and charges the tRNA molecule in trans. Structurally, there are two catalytic domains, referred to as glutamine-recognition (QR) and acyl-transferase (ATRib). We report here the essential catalytic core of the QR domain as determined by extensive biochemical probing, mutation, and structural minimization. The minimal core of the QR domain is a 29-nt helix-loop RNA, which is also able to glutaminylate ATRib in trans. Its amino acid binding site is embedded in an 11-nt cluster that is adjacent to the loop that interacts with the ATRib domain. Our study shows that a minihelix-loop RNA can act as a trans-aminoacylation catalyst, which lends support for the critical role of minihelix-loops in the early evolution of the aminoacylation system.