Critical domain interactions for type A RNase P RNA catalysis with and without the specificity domain.

The natural trans-acting ribozyme RNase P RNA (RPR) is composed of two domains in which the catalytic (C-) domain mediates cleavage of various substrates. The C-domain alone, after removal of the second specificity (S-) domain, catalyzes this reaction as well, albeit with reduced efficiency. Here we provide experimental evidence indicating that efficient cleavage mediated by the Escherichia coli C-domain (Eco CP RPR) with and without the C5 protein likely depends on an interaction referred to as the "P6-mimic". Moreover, the P18 helix connects the C- and S-domains between its loop and the P8 helix in the S-domain (the P8/ P18-interaction). In contrast to the "P6-mimic", the presence of P18 does not contribute to the catalytic performance by the C-domain lacking the S-domain in cleavage of an all ribo model hairpin loop substrate while deletion or disruption of the P8/ P18-interaction in full-size RPR lowers the catalytic efficiency in cleavage of the same model hairpin loop substrate in keeping with previously reported data using precursor tRNAs. Consistent with that P18 is not required for cleavage mediated by the C-domain we show that the archaeal Pyrococcus furiosus RPR C-domain, which lacks the P18 helix, is catalytically active in trans without the S-domain and any protein. Our data also suggest that the S-domain has a larger impact on catalysis for E. coli RPR compared to P. furiosus RPR. Finally, we provide data indicating that the absence of the S-domain and P18, or the P8/ P18-interaction in full-length RPR influences the charge distribution near the cleavage site in the RPR-substrate complex to a small but reproducible extent.

Cleavage mediated by the catalytic domain of bacterial RNase P RNA.

Like other RNA molecules, RNase P RNA (RPR) is composed of domains, and these have different functions. Here, we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model RNA hairpin loop substrates. Compared to full-length Eco RPR, the rate constant, k(obs), of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13,000-fold depending on substrate. Specifically, the structural architecture of the -1/+73 played a significant role where a C(-1)/G(+73) pair had the most dramatic effect on k(obs). Substitution of A(248) (E. coli numbering), positioned near the cleavage site in the RNase P-substrate complex, with G in the CP RPR resulted in 30-fold improvement in rate. In contrast, strengthening the interaction between the RPR and the 3' end of the substrate only had a modest effect. Interestingly, although deleting the S-domain gave a reduction in the rate, it resulted in a less erroneous RPR with respect to cleavage site selection. These data support and extend our understanding of the coupling between the distal interaction between the S-domain and events at the active site. Our findings will also be discussed with respect to the structure of RPR derived from different organisms.

Functional coupling between a distal interaction and the cleavage site in bacterial RNase-P-RNA-mediated cleavage.

Bacterial RNase P consists of one protein and one RNA [RNase P RNA (RPR)]. RPR can process tRNA precursors correctly in the absence of the protein. Here we have used model hairpin loop substrates corresponding to the acceptor, T-stem, and T-loop of a precursor tRNA to study the importance of the T-loop structure in RPR-alone reaction. T-stem/loop (TSL) interacts with a region in RPR [TSL binding site (TBS)], forming TSL/TBS interaction. Altering the T-loop structure affects both cleavage site selection and rate of cleavage at the correct site +1 and at the alternative site -1. The magnitude of variation depended on the structures of the T-loop and the TBS region, with as much as a 150-fold reduction in the rate of cleavage at +1. Interestingly, for one T-loop structure mutant, no difference in the rate at -1 was detected compared to cleavage of the substrate with an unchanged T-loop, indicating that, in this case, the altered T-loop structure primarily influences events required for efficient cleavage at the correct site +1. We also provide data supporting a functional link between a productive TSL/TBS interaction and events at the cleavage site. Collectively, our findings emphasize the interplay between separate regions upon formation of a productive RPR substrate that leads to efficient and accurate cleavage. These new data provide support for an induced-fit mechanism in bacterial RPR-mediated cleavage at the correct site +1.

Mechanism of hcnA mRNA recognition in the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens.

In the plant-beneficial bacterium Pseudomonas fluorescens CHA0, the expression of antifungal exoproducts is controlled by the GacS/GacA two-component system. Two RNA binding proteins (RsmA, RsmE) ensure effective translational repression of exoproduct mRNAs. At high cell population densities, GacA induces three small RNAs (RsmX, RsmY, RsmZ) which sequester both RsmA and RsmE, thereby relieving translational repression. Here we systematically analyse the features that allow the RNA binding proteins to interact strongly with the 5' untranslated leader mRNA of the P. fluorescens hcnA gene (encoding hydrogen cyanide synthase subunit A). We obtained evidence for three major RsmA/RsmE recognition elements in the hcnA leader, based on directed mutagenesis, RsmE footprints and toeprints, and in vivo expression data. Two recognition elements were found in two stem-loop structures whose existence in the 5' leader region was confirmed by lead(II) cleavage analysis. The third recognition element, which overlapped the hcnA Shine-Dalgarno sequence, was postulated to adopt either an open conformation, which would favour ribosome binding, or a stem-loop structure, which may form upon interaction with RsmA/RsmE and would inhibit access of ribosomes. Effective control of hcnA expression by the Gac/Rsm system appears to result from the combination of the three appropriately spaced recognition elements.

Structure probing of tmRNA in distinct stages of trans-translation.

Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helper protein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work we used lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery at different steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reacted with Ala-tmRNA*EF-Tu*GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was probed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we also analyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. We observed some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNA conformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA is in complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.

Lead(II) cleavage analysis of RNase P RNA in vivo.

The overall conformation of M1 RNA, the catalytic RNA subunit of RNase P in Escherichia coli, was analyzed in vivo and, in the presence of the C5 protein subunit, in vitro by lead(II) acetate probing. The partial cleavage patterns obtained are congruent with previous structure mapping performed in vitro. Most of the known major and minor cleavages in M1 RNA were supported and could be mapped onto a secondary structure model. The data obtained indicate that C5 has only minor effects on the overall structure of the RNA subunit. The similar cleavage patterns obtained in vitro and in vivo furthermore suggest that the intracellular environment does not greatly alter the overall conformation of M1 RNA within the holoenzyme complex. Moreover, our data indicate that M1 RNA in vivo is present in at least two states-the major fraction is bound to tRNA substrates and a minor fraction is substrate free. Finally, both in this and previous work we found that lead(II) probing data from in vivo experiments conducted on longer RNAs (tmRNA and M1 RNA) generally gives superior resolution compared to parallel in vitro experiments. This may reflect the absence of alternative conformers present in vitro and the more natural state of these RNAs in the cell due to proper, co-transcriptional folding pathways and possibly the presence of RNA chaperones.

A repeated GGA motif is critical for the activity and stability of the riboregulator RsmY of Pseudomonas fluorescens.

The riboregulator RsmY of Pseudomonas fluorescens strain CHA0 is an example of small regulatory RNAs belonging to the global Rsm/Csr regulatory systems controlling diverse cellular processes such as glycogen accumulation, motility, or formation of extracellular products in various bacteria. By binding multiple molecules of the small regulatory protein RsmA, RsmY relieves the negative effect of RsmA on the translation of several target genes involved in the biocontrol properties of strain CHA0. RsmY and functionally related riboregulators have repeated GGA motifs predicted to be exposed in single-stranded regions, notably in the loops of hairpins. The secondary structure of RsmY was corroborated by in vivo cleavage with lead acetate. RsmY mutants lacking three or five (out of six) of the GGA motifs showed reduced ability to derepress the expression of target genes in vivo and failed to bind the RsmA protein efficiently in vitro. The absence of GGA motifs in RsmY mutants resulted in reduced abundance of these transcripts and in a shorter half-life (< or = 6 min as compared with 27 min for wild type RsmY). These results suggest that both the interaction of RsmY with RsmA and the stability of RsmY strongly depend on the GGA repeats and that the ability of RsmY to interact with small regulatory proteins such as RsmA may protect this RNA from degradation.

Lead(II) as a probe for investigating RNA structure in vivo.

In this communication, we describe a simple and reliable method for RNA structure determination in vivo, using the divalent ion, lead(II), as a structural probe. Lead(II) is known to cleave RNA within single-stranded regions, loops, and bulges, whereas cleavages in double-stranded regions are weaker or absent. Because the ion easily entered bacterial cells, Escherichia coli cultures were treated by addition of 50-100 mM lead(II) acetate for 3-7 min, resulting in partial cleavage of RNA in vivo. Cleavage positions were mapped by reverse transcription analysis of total extracted RNA. Three RNAs were analyzed: tmRNA, CopT (the target of the antisense RNA CopA), and the leader region of the ompF mRNA. All three RNAs had previously been analyzed in vitro, and secondary structure models were available. The results presented here show that lead(II) cleavages in vivo yield detailed structural information for these RNAs, which was in good agreement with the models proposed based on in vitro work. These data illustrate the potential of lead(II) as a sequence-independent RNA structure probe for use in living cells.