Recent bioinformatics-aided searches have identified many new small RNAs (sRNAs) in the intergenic regions of the bacterium Escherichia coli. Here, a shot-gun cloning approach (RNomics) was used to generate cDNA libraries of small sized RNAs. Besides many of the known sRNAs, we found new species that were not predicted previously. The present work brings the number of sRNAs in E.coli to 62. Experimental transcription start site mapping showed that some sRNAs were encoded from independent genes, while others were processed from mRNA leaders or trailers, indicative of a parallel transcriptional output generating sRNAs co-expressed with mRNAs. Two of these RNAs (SroA and SroG) consist of known (THI and RFN) riboswitch elements. We also show that two recently identified sRNAs (RyeB and SraC/RyeA) interact, resulting in RNase III-dependent cleavage. To the best of our knowledge, this represents the first case of two non-coding RNAs interacting by a putative antisense mechanism. In addition, intracellular metabolic stabilities of sRNAs were determined, including ones from previous screens. The wide range of half-lives (<2 to >32 min) indicates that sRNAs cannot generally be assumed to be metabolically stable. The experimental characterization of sRNAs analyzed here suggests that the definition of an sRNA is more complex than previously assumed.

The antisense RNA, CopA, regulates the replication frequency of plasmid R1 through inhibition of RepA translation by rapid and specific binding to its target RNA (CopT). The stable CopA-CopT complex is characterized by a four-way junction structure and a side-by-side alignment of two long intramolecular helices. The significance of this structure for binding in vitro and control in vivo was tested by mutations in both CopA and CopT. High rates of stable complex formation in vitro and efficient inhibition in vivo required initial loop-loop complexes to be rapidly converted to extended interactions. These interactions involve asymmetric helix progression and melting of the upper stems of both RNAs to promote the formation of two intermolecular helices. Data presented here delineate the boundaries of these helices and emphasize the need for unimpeded helix propagation. This process is directional, i.e. one of the two intermolecular helices (B) must form first to allow formation of the other (B'). A binding pathway, characterized by a hierarchy of intermediates leading to an irreversible and inhibitory RNA-RNA complex, is proposed.

Thousands of introns have been localized to rRNA genes throughout the three domains of life. The consequences of the presence of either a spliced or an unspliced intron in a rRNA for ribosome assembly and packaging are largely unknown. To help address these questions, and to begin an intron imaging study, we selected a member of the self-splicing group II intron family, which is hypothesized to be the progenitor not only of spliceosomal introns but also of non-LTR retrotransposons. We cloned the self-splicing group II Ll.LtrB intron from Lactococcus lactis into L. lactis 23S rRNA. The 2,492-nt Ll.LtrB intron comprises a catalytic core and an ORF, which encodes a protein, LtrA. LtrA forms a ribonucleoprotein (RNP) complex with the intron RNA to mediate splicing and mobility. The chimeric 23S-intron RNA was shown to be splicing proficient in its native host in the presence of LtrA. Furthermore, a low-resolution cryo-EM reconstruction of the L. lactis ribosome fused to the intron-LtrA RNP of a splicing-defective Ll.LtrB intron was obtained. The image revealed the intron as a large, well defined structure. The activity and structural integrity of the intron indicate not only that it can coexist with the ribosome but also that its presence permits the assembly of a stable ribosome. Additionally, we view our results as a proof of principle that ribosome chimeras may be generally useful for studying a wide variety of structured RNAs and RNP complexes that are not amenable to NMR, crystallographic, or single-particle cryo-EM methodologies.cryo-EM ͉ ribonucleoprotein complex ͉ ribosome-intron chimera I ntrons are common residents of rRNA genes (rDNAs). The small and large subunit rDNAs can harbor the autocatalytic group I and group II introns, as well as the non-self-splicing, archaeal, and spliceosomal introns (1). The location of introns in the 16S and 23S rRNAs is not random; rather, many introns of different types cluster near tRNA-binding sites. Some of these sites are located at the interface between the small and large subunits of the ribosome (2), in dynamic regions that undergo conformational changes during translation (3). The distribution of these introns suggests that their location could be related to their mode of entry into rDNA or rRNA or to their retention at specific sites. Intron maintenance could be a function of the splicing efficiency at a particular position or of the functional preservation of a ribosome harboring an intron.Besides homologous recombination among rDNA genes, there are two different mechanisms of integration of mobile introns that could explain how introns spread into specific ribosomal sequences: homing into sites on DNA and reverse splicing into sites on RNA (4). For this study, we used a member of the self-splicing group II intron family, which is hypothesized to be the progenitor not only of spliceosomal introns (5) but also of non-LTR retrotransposons (6). Our model intron is the Lactococcus lactis group II intron, Ll.LtrB. In a study of the dispersal of Ll.LtrB by retrotranspo...

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