Mechanism of maturase-promoted group II intron splicing.

Mobile group II introns encode reverse transcriptases that also function as intron-specific splicing factors (maturases). We showed previously that the reverse transcriptase/maturase encoded by the Lactococcus lactis Ll.LtrB intron has a high affinity binding site at the beginning of its own coding region in an idiosyncratic structure, DIVa. Here, we identify potential secondary binding sites in conserved regions of the catalytic core and show via chemical modification experiments that binding of the maturase induces the formation of key tertiary interactions required for RNA splicing. The interaction with conserved as well as idiosyncratic regions explains how maturases in some organisms could evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns.

UV-induced crosslinks in the 16S rRNAs of Escherichia coli, Bacillus subtilis and Thermus aquaticus and their implications for ribosome structure and photochemistry.

Sixteen long-range crosslinks are induced in Escherichia coli 16S rRNA by far-UV irradiation. Crosslinking patterns in two other organisms, Bacillus subtilis and Thermus aquaticus, were investigated to determine if the number and location of crosslinks in E.coli occur because of unusually photoreactive nucleotides at particular locations in the rRNA sequence. Thirteen long-range crosslinks in B.subtilis and 15 long-range crosslinks in T.aquaticus were detected by gel electrophoresis and 10 crosslinks in each organism were identified completely by reverse transcription analysis. Of the 10 identified crosslinks in B.subtilis, eight correspond exactly to E.coli crosslinks and two crosslinks are formed close to sites of crosslinks in E.coli. Of the 10 identified crosslinks in T.aquaticus, five correspond exactly to E.coli crosslinks, three are formed close to E.coli crosslinking sites, one crosslink corresponds to a UV laser irradiation-induced crosslink in E.coli and the last is not seen in E.coli. The overall similarity of crosslink positions in the three organisms suggests that the crosslinks arise from tertiary interactions that are highly conserved but with differences in detail in some regions.

Effects of tetracycline and spectinomycin on the tertiary structure of ribosomal RNA in the Escherichia coli 30 S ribosomal subunit.

Structural analysis of the 16 S rRNA in the 30 S subunit and 70 S ribosome in the presence of ribosome-specific antibiotics was performed to determine whether they produced rRNA structural changes that might provide further insight to their action. An UV cross-linking procedure that determines the pattern and frequency of intramolecular 16 S RNA cross-links was used to detect differences reflecting structural changes. Tetracycline and spectinomycin have specific effects detected by this assay. The presence of tetracycline inhibits the cross-link C967xC1400 completely, increases the frequency of cross-link C1402x1501 twofold, and decreases the cross-link G894xU244 by one-half without affecting other cross-links. Spectinomycin reduces the frequency of the cross-link C934xU1345 by 60% without affecting cross-linking at other sites. The structural changes occur at concentrations at which the antibiotics exert their inhibitory effects. For spectinomycin, the apparent binding site and the affected cross-linking site are distant in the secondary structure but are close in tertiary structure in several recent models, indicating a localized effect. For tetracycline, the apparent binding sites are significantly separated in both the secondary and the three-dimensional structures, suggesting a more regional effect.

Dependence of the 16S rRNA decoding region structure on Mg2+, subunit association, and temperature.

The effects of Mg2+ concentration, subunit association, and temperature on the structure of 16S rRNA in the Escherichia coli ribosome were investigated using UV cross-linking and gel electrophoresis analysis. Mg2+ concentrations between 1 and 20 mM and temperatures between 5 and 55 degreesC had little effect on the frequency of 12 of the 14 cross-links in 30S subunits and modest effects on the same cross-links in 70S ribosomes. In contrast, two cross-links, C967 x C1400 and C1402 x C1501, involving rRNA in the decoding region are present in 30S subunits only above 3 mM Mg2+, increase in frequency at higher Mg2+ concentration, and are both more frequent when 50S subunits are included in the reactions. In 70S ribosomes, the cross-link C1402 x C1501 increases but the cross-link C967 x C1400 decreases at higher Mg2+ concentrations. One cross-link, C1397 x U1495, is detected only in 70S ribosomes and decreases in frequency as Mg2+ concentration is increased. An additional cross-link, A1093 x C1182, decreases upon subunit association. The cross-link frequency differences indicate that the arrangement of the decoding region of the 16S rRNA, but not in the rest of the subunit, is readily altered by Mg2+ ions and subunit association.

Exact determination of UV-induced crosslinks in 16S ribosomal RNA in 30S ribosomal subunits.

Escherichia coli 30S ribosomal subunits were UV-irradiated to induce intramolecular crosslinks in the 16S rRNA. Intact 16S rRNA was purified and subjected to gel electrophoresis, under denaturing conditions, to separate molecules on the basis of the crosslinked loop size. Molecules separated this way were enriched for specific crosslinks and could be analyzed by the reverse transcription arrest assay to determine exact crosslinking sites. Thirteen crosslinking sites have been identified at single nucleotide resolution. Of these, eight are within or adjacent to secondary structure elements: one of these (C582 x G760) involves an interaction between nucleotides within an interior loop, one (C1402 x X1501) involves an interaction between nucleosides in adjacent base pairs, and the others involve interactions between nucleotides that are within junction regions (A441 x G494, U562 x U884, C934 x U1345, and U991 x U1212) or are interactions between nucleotides (C54 x A353 and U1052 x C1200) that somehow cross known base pairs. Five other crosslinks connect sites distant in the secondary structure and provide global constraints for the arrangement of RNA regions within RNA domains I and II (U244 x G894, G894 x A1468, C967 x C1400) and within domain III (U1126 x C1281 and A1093 x G1182). These crosslinks, known at single-nucleotide resolution, are useful in the prediction of local RNA regions, as well as the global structure.