Small RNA sequences are readily stabilized by inclusion in a carrier rRNA.

This laboratory previously showed that an RNA derived from 5S ribosomal RNA could be used as a carrier to harbor a nucleic acid "tag" for monitoring genetically engineered or naturally occurring bacteria. The prototype system expressed a specific tagged RNA that was stable and accumulated to high levels. For such a system to be useful there should, however, be little limitation on the sequence composition and length of the insert. To test these limitations, a collection of insertion sequences were created and introduced into the artificial 5S rRNA cassette. This library consisted of random 13- and 50-base oligonucleotides that were inserted into the carrier RNA. We report here that essentially all of the insert-containing RNAs are stable and accumulate to detectable levels. Tagged RNAs were produced by both plasmid-borne and chromosomally integrated expression systems in E. coli and several Pseudomonas strains without obvious effect on the host cell. It is anticipated that in addition to its intended use in environmental monitoring, this system can be used for in vivo selection of useful artificial RNAs. Because the carrier lends stability to the RNAs, the system may also be useful in RNA production.

Common 5S rRNA variants are likely to be accepted in many sequence contexts.

Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The results demonstrate that changes that occur multiple times in a local region of RNA sequence space in fact usually will be accepted in any sequence context in that same local region.

Mutations in the Lactococcus lactis Ll.LtrB group II intron that retain mobility in vivo.

BACKGROUND: Group II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron (Ll.LtrB) and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested. RESULTS: The largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb(1-2). This mutant had a mobility frequency comparable to the wild-type Ll.LtrB intron (DeltaORF construct). Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II (nts 404-459) was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent. CONCLUSIONS: The smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb(1-2) and DII(ii)a,b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.

Binding of a group II intron-encoded reverse transcriptase/maturase to its high affinity intron RNA binding site involves sequence-specific recognition and autoregulates translation.

Mobile group II introns encode reverse transcriptases that bind specifically to the intron RNAs to promote both intron mobility and RNA splicing (maturase activity). Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein (LtrA) binds first to a primary high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of the LtrA coding sequence, and then makes additional contacts with conserved regions of the intron to fold the RNA into the catalytically active structure. Here, we analyzed the DIVa binding site by iterative in vitro selection and in vitro mutagenesis. Our results show that LtrA binds to a small region at the distal end of DIVa that contains the ribosome-binding site and initiation codon of the LtrA open reading frame. The critical elements are in a small stem-loop structure emanating from a purine-rich internal loop, with both sequence and structure playing a role in LtrA recognition. The ribosome-binding site falls squarely within the LtrA-binding region and is sequestered directly by the binding of LtrA or by stabilization of the small stem-loop or both. Finally, by using LacZ fusions in Escherichia coli, we show that the binding of LtrA to DIVa down-regulates translation. This mode of regulation limits accumulation of the potentially deleterious intron-encoded protein and may facilitate splicing by halting ribosome entry into the intron. The recognition of the DIVa loop-stem-loop structure accounts, in part, for the intron specificity of group II intron maturases and has parallels in template-recognition mechanisms used by other reverse transcriptases.

The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs.

BACKGROUND: Comparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-organized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields. RESULTS: We have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems. CONCLUSIONS: This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site http://www.rna.icmb.utexas.edu. In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site.