Efficient RNA pseudouridylation by eukaryotic H/ACA ribonucleoproteins requires high affinity binding and correct positioning of guide RNA.

H/ACA ribonucleoproteins (H/ACA RNPs) are responsible for introducing many pseudouridines into RNAs, but are also involved in other cellular functions. Utilizing a purified and reconstituted yeast H/ACA RNP system that is active in pseudouridine formation under physiological conditions, we describe here the quantitative characterization of H/ACA RNP formation and function. This analysis reveals a surprisingly tight interaction of H/ACA guide RNA with the Cbf5p-Nop10p-Gar1p trimeric protein complex whereas Nhp2p binds comparably weakly to H/ACA guide RNA. Substrate RNA is bound to H/ACA RNPs with nanomolar affinity which correlates with the GC content in the guide-substrate RNA base pairing. Both Nhp2p and the conserved Box ACA element in guide RNA are required for efficient pseudouridine formation, but not for guide RNA or substrate RNA binding. These results suggest that Nhp2p and the Box ACA motif indirectly facilitate loading of the substrate RNA in the catalytic site of Cbf5p by correctly positioning the upper and lower parts of the H/ACA guide RNA on the H/ACA proteins. In summary, this study provides detailed insight into the molecular mechanism of H/ACA RNPs.

Taking a Step Back from Back-Translocation: an Integrative View of LepA/EF4's Cellular Function.

Protein synthesis, the translation of mRNA into a polypeptide facilitated by the ribosome, is assisted by a variety of protein factors, some of which are GTPases. In addition to four highly conserved and well-understood GTPases with known function, there are also a number of noncanonical GTPases that are implicated in translation but whose functions are not fully understood. LepA/EF4 is one of these noncanonical GTPases. It is highly conserved and present in bacteria, mitochondria, and chloroplasts, but its functional role in the cell remains unknown. LepA's sequence and domain arrangement are very similar to those of other translational GTPases, but it contains a unique C-terminal domain (CTD) that is likely essential to its specific function in the cell. Three main hypotheses about the function of LepA have been brought forward to date: (i) LepA is a back-translocase, (ii) LepA relieves ribosome stalling or facilitates sequestration, and (iii) LepA is involved in ribosome biogenesis. This review examines the structural and biochemical information available on bacterial LepA and discusses it on the background of the available in vivo information from higher organisms in order to broaden the view regarding LepA's functional role in the cell and how the structure of its unique CTD might be involved in facilitating this role.

tRNA binding, positioning, and modification by the pseudouridine synthase Pus10.

Pus10 is the most recently identified pseudouridine synthase found in archaea and higher eukaryotes. It modifies uridine 55 in the TΨC arm of tRNAs. Here, we report the first quantitative biochemical analysis of tRNA binding and pseudouridine formation by Pyrococcus furiosus Pus10. The affinity of Pus10 for both substrate and product tRNA is high (Kd of 30nM), and product formation occurs with a Km of 400nM and a kcat of 0.9s(-1). Site-directed mutagenesis was used to demonstrate that the thumb loop in the catalytic domain is important for efficient catalysis; we propose that the thumb loop positions the tRNA within the active site. Furthermore, a new catalytic arginine residue was identified (arginine 208), which is likely responsible for triggering flipping of the target uridine into the active site of Pus10. Lastly, our data support the proposal that the THUMP-containing domain, found in the N-terminus of Pus10, contributes to binding of tRNA. Together, our findings are consistent with the hypothesis that tRNA binding by Pus10 occurs through an induced-fit mechanism, which is a prerequisite for efficient pseudouridine formation.

Archaeal proteins Nop10 and Gar1 increase the catalytic activity of Cbf5 in pseudouridylating tRNA.

Cbf5 is a pseudouridine synthase that usually acts in a guide RNA-dependent manner as part of H/ACA small ribonucleoproteins; however archaeal Cbf5 can also act independently of guide RNA in modifying uridine 55 in tRNA. This guide-independent activity of Cbf5 is enhanced by proteins Nop10 and Gar1 which are also found in H/ACA small ribonucleoproteins. Here, we analyzed the specific contribution of Nop10 and Gar1 for Cbf5-catalyzed pseudouridylation of tRNA. Interestingly, both Nop10 and Gar1 not only increase Cbf5's affinity for tRNA, but they also directly enhance Cbf5's catalytic activity by increasing the k(cat) of the reaction. In contrast to the guide RNA-dependent reaction, Gar1 is not involved in product release after tRNA modification. These results in conjunction with structural information suggest that Nop10 and Gar1 stabilize Cbf5 in its active conformation; we hypothesize that this might also be true for guide-RNA dependent pseudouridine formation by Cbf5.