Insights into RNase P RNA structure and function by a retro-evolution approach
RNase P catalyzes tRNA 5’-end maturation in all organisms and organelles. The enzyme is composed of a single RNA subunit plus a number of proteins that increases from bacteria (one protein) over archaea (at least four proteins) to eukarya (nine to ten proteins). Conserved base identities indicate th...
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| Format: | Dissertation |
| Sprache: | Englisch |
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Philipps-Universität Marburg
2009
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| Zusammenfassung: | RNase P catalyzes tRNA 5’-end maturation in all organisms and organelles. The enzyme is composed of a single RNA subunit plus a number of proteins that increases from bacteria (one protein) over archaea (at least four proteins) to eukarya (nine to ten proteins). Conserved base identities indicate that the RNase P RNA subunits (P RNAs) from all three kingsdoms of life stem from a common ancestor. Yet, only in bacteria the P RNA alone is substantially active in vitro without the protein, whereas archaeal and eukaryal P RNAs are more dependent on the contribution of their protein moieties and display only residual activity when these are absent. RNase P thus represents a natural model system for the switch from ribozyme to ribonucleoprotein enzyme, generally accepted to have occurred during natural evolution of the RNA world to the protein world.
The RNA subunit of cyanelle RNase P from Cyanophora paradoxa contains essentially all structural elements of bacterial P RNAs, but has been reported to be catalytically inactive. In contrast to these previous observations, we were able to detect activity of this P RNA in the absence of protein cofactors. Furthermore, the C. paradoxa P RNA forms a functional holoenzyme with proteobacterial P proteins. Analysis of C- and S-domain swaps between cyanelle and Escherichia coli P RNA revealed that their domains have the capacity to cooperate, because the hybrid RNAs were functional. However, activities of the chimeras lagged behind the catalytic performance of the bacterial P RNA, suggesting that both the C- and S-domain of the cyanelle P RNA are weakly functional, thus limiting the activity of each type of chimera (EC or CE). In addition or alternatively, domain interaction and overall folding may be suboptimal in the chimeras, as it likely is in the wild-type cyanelle P RNA. Furthermore, the organellar ribozyme is unusual compared to the consensus of bacterial P RNAs: RNA-alone activity is low and structural alterations as small as point mutations or switches in Watson-Crick base pair identity at positions that are not part of the universally conserved catalytic core abrogate activity as does incorporating the E. coli L15-16 loop and adjacent regions. These findings indicate that the A,U-rich cyanelle RNase P RNA is globally optimized but conformationally unstable, thus representing an RNase P type of its own rather than simply being a slightly degenerate form of bacterial RNase P. In this context the E. coli P protein nevertheless emerged as a universal player in P RNA activation, and it will be all the more interesting to see if bacterial P proteins are related to any of the P protein components specific to the C. paradoxa cyanelle that yet await identification.
The increased protein proportion of archaeal and eukaryal RNase P holoenzymes parallels a vast decrease in the catalytic activity of their RNA subunits (P RNAs) alone. We show that a few mutations toward the bacterial P RNA consensus substantially activate the C-domain of archaeal P RNA from M. thermoautotrophicus, in the absence and presence of the bacterial RNase P protein. Large increases in ribozyme activity required the cooperative effect of at least two structural alterations. The P1 helix of P RNA from M. thermoautotrophicus was found to be extended, which increases ribozyme activity (ca 200-fold) and stabilizes the tertiary structure. Activity increases of mutated archaeal C-domain variants were more pronounced in the context of chimeric P RNAs carrying the bacterial S-domain of Escherichia coli instead of the archaeal S-domain. This could be explained by the loss of the archaeal S-domain’s capacity to support tight and productive substrate binding in the absence of protein cofactors. Our results demonstrate that the catalytic capacity of archaeal P RNAs is close to that of their bacterial counterparts, but is masked by minor changes in the C-domain and, particularly, by poor function of the archaeal S-domain in the absence of archaeal protein cofactors.
The eukaryal RNase P RNAs from human (H1 RNA) and the lower eukarya Giardia lamblia were recently found to have residual catalytic activity. Nevertheless, the highest activities measured for these eukaryotic P RNAs are more than five orders of magnitude lower than those obtained with bacterial P RNAs. To investigate the structure and function of H1 RNA, we analyzed if the RNA-lone activity of H1 RNA could be improved by approaches that have proven successful in the activation of M. thermoautotrophicus archaeal RNase P RNA. A few H1 RNA variants, HE chimeras and H1 RNA-substrate conjugates were constructed and analyzed. H1 RNA 5, only containing mutations P4/J4/19, is the sole variant with locally confined mutations that showed increased activity. The mutations P4/J4/19 were introduced to restore a bacterial-like P4 known to be a crucial for catalytic activity. The additionally introduced back-to-bacteria mutations P5/P15 further improved catalytic activity slightly, possibly by rigidifying the core structure and/or improving substrate binding. Furthermore, the S-domain of E. coli is unable to productively cooperate with the H1 RNA C-domain, as the chimeric P RNA HE was found to be inactive. Tethering the substrate to H1 RNA, designed to mitigate weak substrate affinity, even reduced H1 RNA activity to non-detectable levels. The kinetic data together with our structural probing and UV melting (not shown) data suggest that H1 RNA has an extremely degenerated, globally unstable structure that is strictly dependent on its natural protein cofactors. |
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| Umfang: | 168 Seiten |
| DOI: | 10.17192/z2009.0124 |