The pistol RNA motif represents a new class of self-cleaving ribozymes of yet unknown biological function. Our recent crystal structure of a pre-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53-U54 cleavage site. While the N1 of G40 is within 3.4 Å of the modeled G53 2'-OH group that attacks the scissile phosphate, thus suggesting a direct role in general acid-base catalysis, the function of A32 is less clear. We present evidence from atom-specific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis. By contrast, the ribose 2'-OH of A32 seems crucial for the proper positioning of G40 through a H-bond network that involves G42 as a bridging unit between A32 and G40. We also found that disruption of the inner-sphere coordination of the active-site Mg cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg playing a catalytic role in cleavage.

New RNA modifications are needed to advance our toolbox for targeted manipulation of RNA. In particular, the development of high-performance reporter groups facilitating spectroscopic analysis of RNA structure and dynamics,...

The pistol RNAmotif represents anew class of selfcleaving ribozymes of yet unknown biological function. Our recent crystal structure of ap re-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53-U54 cleavage site.W hile the N1 of G40 is within 3.4 of the modeled G53 2'-OH group that attacks the scissile phosphate, thus suggesting adirect role in general acid-base catalysis,the function of A32 is less clear.W ep resent evidence from atomspecific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis.Bycontrast, the ribose 2'-OH of A32 seems crucial for the proper positioning of G40 through aH-bond network that involves G42 as abridging unit between A32 and G40. We also found that disruption of the innersphere coordination of the active-site Mg 2+ cation to N7 of G33 makes the ribozyme drastically slower.Amechanistic proposal is suggested, with A32 playing as tructural role and hydrated Mg 2+ playing ac atalytic role in cleavage.

Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.

Post-transcriptional modifications are added to ribosomal RNAs (rRNAs) to govern ribosome biogenesis and to fine-tune protein biosynthesis. In Escherichia coli and related bacteria, RlhA uniquely catalyzes formation of a 5-hydroxycytidine (ho5C) at position 2501 of 23S rRNA. However, the molecular and biological functions as well as the regulation of ho5C2501 modification remain unclear. We measured growth curves with the modification-deficient ΔrlhA strain and quantified the extent of the modification during different conditions by mass spectrometry and reverse transcription. The levels of ho5C2501 in E. coli ribosomes turned out to be highly dynamic and growth phase-dependent, with the most effective hydroxylation yields observed in the stationary phase. We demonstrated a direct effect of ho5C2501 on translation efficiencies in vitro and in vivo. High ho5C2501 levels reduced protein biosynthesis which however turned out to be beneficial for E. coli for adapting to oxidative stress. This functional advantage was small under optimal conditions or during heat or cold shock, but becomes pronounced in the presence of hydrogen peroxide. Taken together, these data provided first functional insights into the role of this unique 23S rRNA modification for ribosome functions and bacterial growth under oxidative stress.