Pseudouridine Synthase RsuA Confers a Survival Advantage to Bacteria under Streptomycin Stress.

Bacterial ribosome small subunit rRNA (16S rRNA) contains 11 nucleotide modifications scattered throughout all its domains. The 16S rRNA pseudouridylation enzyme, RsuA, which modifies U516, is a survival protein essential for bacterial survival under stress conditions. A comparison of the growth curves of wildtype and RsuA knock-out E. coli strains illustrates that RsuA renders a survival advantage to bacteria under streptomycin stress. The RsuA-dependent growth advantage for bacteria was found to be dependent on its pseudouridylation activity. In addition, the role of RsuA as a trans-acting factor during ribosome biogenesis may also play a role in bacterial growth under streptomycin stress. Furthermore, circular dichroism spectroscopy measurements and RNase footprinting studies have demonstrated that pseudouridine at position 516 influences helix 18 structure, folding, and streptomycin binding. This study exemplifies the importance of bacterial rRNA modification enzymes during environmental stress.

Peptides Targeting RNA m6 A Methylations Influence the Viability of Cancer Cells.

N6-methyladenosine (m6 A) is the most abundant nucleotide modification observed in eukaryotic mRNA. Changes in m6 A levels in transcriptome are tightly correlated to expression levels of m6 A methyltransferases and demethylases. Abnormal expression levels of methyltransferases and demethylases are observed in various diseases and health conditions such as cancer, male infertility, and obesity. This research explores the efficacy of m6 A-modified RNA as an anticancer drug target. We discovered a 12-mer peptide that binds specifically to m6 A-modified RNA using phage display experiments. Our fluorescence-based assays illustrate the selected peptide binds to methylated RNA with lower micromolar affinity and inhibit the binding of protein FTO, a demethylase enzyme specific to m6 A modification. When cancer cell lines were treated with mtp1, it led to an increase in m6 A levels and a decrease in cell viability. Hence our results illustrate the potential of mtp1 to be developed as a drug for cancer.

RsmG forms stable complexes with premature small subunit rRNA during bacterial ribosome biogenesis.

The ribosome is the ribonucleoprotein machine that carries out protein biosynthesis in all forms of life. Perfect synchronization between ribosomal RNA (rRNA) transcription, folding, post-transcriptional modification, maturation, and assembly of r-proteins is essential for the rapid formation of structurally and functionally accurate ribosomes. Many RNA nucleotide modification enzymes may function as assembly factors that oversee the accuracy of ribosome assembly. The protein RsmG is a methyltransferase enzyme that is responsible for N7 methylation in G527 of 16S rRNA. Here we illustrate the ability of RsmG to bind various premature small subunit ribosomal RNAs with contrasting affinities. Protein RsmG binds with approximately 15-times higher affinity to premature 16S rRNA with the full leader sequence compared to that of mature 16S rRNA. Various r-proteins which bind to the 5'-domain influence RsmG binding. The observed binding cooperativity between RsmG and r-proteins is sensitive to the maturation status of premature small subunit rRNA. However, neither the maturation of 16S rRNA nor the presence of various r-proteins significantly influence the methylation activity of RsmG. The capability of RsmG to bind to premature small subunit rRNA and alter its binding preference to various RNA-protein complexes based on the maturation of rRNA indicates its ability to influence ribosome assembly.

A metastable rRNA junction essential for bacterial 30S biogenesis.

Tertiary sequence motifs encode interactions between RNA helices that create the three-dimensional structures of ribosomal subunits. A Right Angle motif at the junction between 16S helices 5 and 6 (J5/6) is universally conserved amongst small subunit rRNAs and forms a stable right angle in minimal RNAs. J5/6 does not form a right angle in the mature ribosome, suggesting that this motif encodes a metastable structure needed for ribosome biogenesis. In this study, J5/6 mutations block 30S ribosome assembly and 16S maturation in Escherichia coli. Folding assays and in-cell X-ray footprinting showed that J5/6 mutations favor an assembly intermediate of the 16S 5' domain and prevent formation of the central pseudoknot. Quantitative mass spectrometry revealed that mutant pre-30S ribosomes lack protein uS12 and are depleted in proteins uS5 and uS2. Together, these results show that impaired folding of the J5/6 right angle prevents the establishment of inter-domain interactions, resulting in global collapse of the 30S structure observed in electron micrographs of mutant pre-30S ribosomes. We propose that the J5/6 motif is part of a spine of RNA helices that switch conformation at distinct stages of assembly, linking peripheral domains with the 30S active site to ensure the integrity of 30S biogenesis.

Evolution of protein-coupled RNA dynamics during hierarchical assembly of ribosomal complexes.

Assembly of 30S ribosomes involves the hierarchical addition of ribosomal proteins that progressively stabilize the folded 16S rRNA. Here, we use three-color single molecule FRET to show how combinations of ribosomal proteins uS4, uS17 and bS20 in the 16S 5' domain enable the recruitment of protein bS16, the next protein to join the complex. Analysis of real-time bS16 binding events shows that bS16 binds both native and non-native forms of the rRNA. The native rRNA conformation is increasingly favored after bS16 binds, explaining how bS16 drives later steps of 30S assembly. Chemical footprinting and molecular dynamics simulations show that each ribosomal protein switches the 16S conformation and dampens fluctuations at the interface between rRNA subdomains where bS16 binds. The results suggest that specific protein-induced changes in the rRNA dynamics underlie the hierarchy of 30S assembly and simplify the search for the native ribosome structure.Ribosomes assemble through the hierarchical addition of proteins to a ribosomal RNA scaffold. Here the authors use three-color single-molecule FRET to show how the dynamics of the rRNA dictate the order in which multiple proteins assemble on the 5' domain of the E. coli 16S rRNA.

Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5'-domain assembly.

Ribosomal protein S4 nucleates assembly of the 30S ribosome 5' and central domains, which is crucial for the survival of cells. Protein S4 changes the structure of its 16S rRNA binding site, passing through a non-native intermediate complex before forming native S4-rRNA contacts. Ensemble FRET was used to measure the thermodynamic stability of non-native and native S4 complexes in the presence of Mg(2+) ions and other 5'-domain proteins. Equilibrium titrations of Cy3-labeled 5'-domain RNA with Cy5-labeled protein S4 showed that Mg(2+) ions preferentially stabilize the native S4-rRNA complex. In contrast, ribosomal proteins S20 and S16 act by destabilizing the non-native S4-rRNA complex. The full cooperative switch to the native complex requires S4, S16, and S20 and is achieved to a lesser degree by S4 and S16. The resulting thermodynamic model for assembly of the 30S body illustrates how ribosomal proteins selectively bias the equilibrium between alternative rRNA conformations, increasing the cooperativity of rRNA folding beyond what can be achieved by Mg(2+) ions alone.

An improved surface passivation method for single-molecule studies.

We report a surface passivation method based on dichlorodimethylsilane (DDS)-Tween-20 for in vitro single-molecule studies, which, under the conditions tested here, more efficiently prevented nonspecific binding of biomolecules than the standard poly(ethylene glycol) surface. The DDS-Tween-20 surface was simple and inexpensive to prepare and did not perturb the behavior and activities of tethered biomolecules. It can also be used for single-molecule imaging in the presence of high concentrations of labeled species in solution.

Pseudouridines in rRNA helix 69 play a role in loop stacking interactions.

The (1)H NMR spectra of RNAs representing E. coli 23S rRNA helix 69 with [1,3-(15)N]pseudouridine modification at specific sites reveal unique roles for pseudouridine in stabilizing base-stacking interactions in the hairpin loop region.

pH-dependent structural changes of helix 69 from Escherichia coli 23S ribosomal RNA.

Helix 69 in 23S rRNA is a region in the ribosome that participates in a considerable number of RNA-RNA and RNA-protein interactions. Conformational flexibility is essential for such a region to interact and accommodate protein factors at different stages of protein biosynthesis. In this study, pH-dependent structural and stability changes were observed for helix 69 through a variety of spectroscopic techniques, such as circular dichroism spectroscopy, UV melting, and nuclear magnetic resonance spectroscopy. In Escherichia coli 23S rRNA, helix 69 contains pseudouridine residues at positions 1911, 1915, and 1917. The presence of these pseudouridines was found to be essential for the pH-induced conformational changes. Some of the pH-dependent changes appear to be localized to the loop region of helix 69, emphasizing the importance of the highly conserved nature of residues in this region.