Periscope Proteins are variable-length regulators of bacterial cell surface interactions.

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

Structures of B. subtilis Maturation RNases Captured on 50S Ribosome with Pre-rRNAs.

The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.

Functional diversification in the Nudix hydrolase gene family drives sesquiterpene biosynthesis in Rosa × wichurana.

Roses use a non-canonical pathway involving a Nudix hydrolase, RhNUDX1, to synthesize their monoterpenes, especially geraniol. Here we report the characterization of another expressed NUDX1 gene from the rose cultivar Rosa x wichurana, RwNUDX1-2. In order to study the function of the RwNUDX1-2 protein, we analyzed the volatile profiles of an F1 progeny generated by crossing R. chinensis cv. 'Old Blush' with R. x wichurana. A correlation test of the volatilomes with gene expression data revealed that RwNUDX1-2 is involved in the biosynthesis of a group of sesquiterpenoids, especially E,E-farnesol, in addition to other sesquiterpenes. In vitro enzyme assays and heterologous in planta functional characterization of the RwNUDX1-2 gene corroborated this result. A quantitative trait locus (QTL) analysis was performed using the data of E,E-farnesol contents in the progeny and a genetic map was constructed based on gene markers. The RwNUDX1-2 gene co-localized with the QTL for E,E-farnesol content, thereby confirming its function in sesquiterpenoid biosynthesis in R. x wichurana. Finally, in order to understand the structural bases for the substrate specificity of rose NUDX proteins, the RhNUDX1 protein was crystallized, and its structure was refined to 1.7 Å. By molecular modeling of different rose NUDX1 protein complexes with their respective substrates, a structural basis for substrate discrimination by rose NUDX1 proteins is proposed.

Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition.

1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson-Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson-Crick R13-A22 base-pair positioning the A22 N1-atom close to the SAM methyl group.

Design of cross-linked RNA/protein complexes for structural studies.

Crystallographic studies of RNA/protein complexes are primordial for the understanding of recognition determinants and catalytic mechanisms in the case of enzymes. However, due to the flexibility and propensity to conformational heterogeneity of RNAs, as well as the mostly electrostatic interactions of RNA/protein complexes, they are difficult to crystallize. We present here a method to trap the two interacting partners in a covalent complex, based on a modified reactive RNA allowing the use of the full range of common crystallogenesis tools. We demonstrate the practicability of our approach with the production of a covalent complex of the Thermus thermophilus m1A58 tRNA modification enzyme, and a modified stem loop mimicking the natural substrate of the enzyme.

Conserved asymmetry underpins homodimerization of Dicer-associated double-stranded RNA-binding proteins.

Double-stranded RNA-binding domains (dsRBDs) are commonly found in modular proteins that interact with RNA. Two varieties of dsRBD exist: canonical Type A dsRBDs interact with dsRNA, while non-canonical Type B dsRBDs lack RNA-binding residues and instead interact with other proteins. In higher eukaryotes, the microRNA biogenesis enzyme Dicer forms a 1:1 association with a dsRNA-binding protein (dsRBP). Human Dicer associates with HIV TAR RNA-binding protein (TRBP) or protein activator of PKR (PACT), while Drosophila Dicer-1 associates with Loquacious (Loqs). In each case, the interaction involves a region of the protein that contains a Type B dsRBD. All three dsRBPs are reported to homodimerize, with the Dicer-binding region implicated in self-association. We report that these dsRBD homodimers display structural asymmetry and that this unusual self-association mechanism is conserved from flies to humans. We show that the core dsRBD is sufficient for homodimerization and that mutation of a conserved leucine residue abolishes self-association. We attribute differences in the self-association properties of Loqs, TRBP and PACT to divergence of the composition of the homodimerization interface. Modifications that make TRBP more like PACT enhance self-association. These data are examined in the context of miRNA biogenesis and the protein/protein interaction properties of Type B dsRBDs.

m1A Post-Transcriptional Modification in tRNAs.

To date, about 90 post-transcriptional modifications have been reported in tRNA expanding their chemical and functional diversity. Methylation is the most frequent post-transcriptional tRNA modification that can occur on almost all nitrogen sites of the nucleobases, on the C5 atom of pyrimidines, on the C2 and C8 atoms of adenosine and, additionally, on the oxygen of the ribose 2'-OH. The methylation on the N1 atom of adenosine to form 1-methyladenosine (m1A) has been identified at nucleotide position 9, 14, 22, 57, and 58 in different tRNAs. In some cases, these modifications have been shown to increase tRNA structural stability and induce correct tRNA folding. This review provides an overview of the currently known m1A modifications, the different m1A modification sites, the biological role of each modification, and the enzyme responsible for each methylation in different species. The review further describes, in detail, two enzyme families responsible for formation of m1A at nucleotide position 9 and 58 in tRNA with a focus on the tRNA binding, m1A mechanism, protein domain organisation and overall structures.

Backbone resonance assignments of the m1A22 tRNA methyltransferase TrmK from Bacillus subtilis.

RNA modification is a post-transcriptional process by which certain nucleotides are altered after their initial incorporation into an RNA chain. Transfer RNAs (tRNAs) is the most heavily modified class of RNA molecules. These modifications expand the chemical and functional diversity of tRNAs and enhance their structural stability. To date, more than 100 modifications have been identified, the majority of which are specific from one domain of life. However, few modifications are extensively present in the three domains of life. Among those, the m(1)A nucleotide, which consists in the methylation at position 1 of the adenine aromatic ring, is found in tRNAs and ribosomal RNAs. In tRNAs, the m(1)A modification occurs at position 9, 14, 22, 57 and 58. The enzyme TrmK catalyzes the m(1)A formation at position 22. Here we report the backbone (1)H, (15)N and (13)C chemical shift assignments of TrmK from Bacillus subtilis obtained by heteronuclear multidimensional NMR spectroscopy as well as its secondary structure in solution as predicted by TALOS+. These assignments of TrmK pave the way for interaction studies with its tRNA substrates.

In Vitro/In Vivo Production of tRNA for X-Ray Studies.

tRNAs occupy a central role in the cellular life, and they are involved in a broad range of biological processes that relies on their interaction with proteins and RNA. Crystallization and structure resolution of tRNA or/and tRNA/partner complexes can yield in valuable information on structural organizations of key elements of cellular machinery. However, crystallization of RNA, is often challenging. Here we review two methods to produce and purify tRNA in quantity and quality to perform X-ray studies.

The m1A(58) modification in eubacterial tRNA: An overview of tRNA recognition and mechanism of catalysis by TrmI.

The enzymes of the TrmI family catalyze the formation of the m(1)A58 modification in tRNA. We previously solved the crystal structure of the Thermus thermophilus enzyme and conducted a biophysical study to characterize the interaction between TrmI and tRNA. TrmI enzymes are active as a tetramer and up to two tRNAs can bind to TrmI simultaneously. In this paper, we present the structures of two TrmI mutants (D170A and Y78A). These residues are conserved in the active site of TrmIs and their mutations result in a dramatic alteration of TrmI activity. Both structures of TrmI mutants revealed the flexibility of the N-terminal domain that is probably important to bind tRNA. The structure of TrmI Y78A catalytic domain is unmodified regarding the binding of the SAM co-factor and the conformation of residues potentially interacting with the substrate adenine. This structure reinforces the previously proposed role of Y78, i.e. stabilize the conformation of the A58 ribose needed to hold the adenosine in the active site. The structure of the D170A mutant shows a flexible active site with one loop occupying in part the place of the co-factor and the second loop moving at the entrance to the active site. This structure and recent data confirms the central role of D170 residue binding the amino moiety of SAM and the exocyclic amino group of adenine. Possible mechanisms for methyl transfer are then discussed.