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1.  BswR controls bacterial motility and biofilm formation in Pseudomonas aeruginosa through modulation of the small RNA rsmZ 
Nucleic Acids Research  2014;42(7):4563-4576.
Pseudomonas aeruginosa relies on cell motility and ability to form biofilms to establish infections; however, the mechanism of regulation remains obscure. Here we report that BswR, a xenobiotic response element-type transcriptional regulator, plays a critical role in regulation of bacterial motility and biofilm formation in P. aeruginosa. Transcriptomic and biochemical analyses showed that BswR counteracts the repressor activity of MvaT, controls the transcription of small RNA rsmZ and regulates the biogenesis of bacterial flagella. The crystal structure of BswR was determined at 2.3 Å resolution; the monomer comprises a DNA-binding domain with a helix-turn-helix motif in the N terminus and two helices (α6 and α7) with a V-shaped arrangement in the C-terminus. In addition to the contacts between the parallel helices α5 of two monomers, the two helical extensions (α6 and α7) intertwine together to form a homodimer, which is the biological function unit. Based on the result of DNase I protection assay together with structural analysis of BswR homodimer, we proposed a BswR–DNA model, which suggests a molecular mechanism with which BswR could interact with DNA. Taken together, our results unveiled a novel regulatory mechanism, in which BswR controls the motility and biofilm formation of P. aeruginosa by modulating the transcription of small RNA rsmZ.
PMCID: PMC3985676  PMID: 24497189
2.  The structure of the ribosome with elongation factor G trapped in the post-translocational state 
Science (New York, N.Y.)  2009;326(5953):694-699.
Elongation factor G (EF-G) is a GTPase that plays a crucial role in the translocation of tRNAs and mRNA during translation by the ribosome. We report a crystal structure refined to 3.6 Å resolution of the ribosome trapped with EF-G in the post-translocational state using the antibiotic fusidic acid. Fusidic acid traps EF-G in a conformation intermediate between the GTP and GDP forms. The interaction of EF-G with ribosomal elements implicated in stimulating catalysis, such as the L10-L12 stalk and the L11 region, and of domain IV of EF-G with P-site tRNA and mRNA shed light on various aspects of EF-G function in catalysis and translocation. The stabilization of the mobile stalks of the ribosome also results in a more complete description of its structure.
PMCID: PMC3763468  PMID: 19833919
3.  The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA 
Science (New York, N.Y.)  2009;326(5953):688-694.
The ribosome selects a correct tRNA for each amino acid added to the polypeptide chain, as directed by mRNA. Aminoacyl-tRNA is delivered to the ribosome by Elongation Factor-Tu (EF-Tu), which hydrolyzes GTP and releases tRNA in response to codon recognition. The signaling pathway that leads to GTP hydrolysis upon codon recognition is critical to accurate decoding. Here we present the crystal structure of the ribosome complexed with EF-Tu and aminoacyl-tRNA, refined to 3.6 Å resolution. The structure reveals details of the tRNA distortion that allows aminoacyl-tRNA to interact simultaneously with the decoding center of the 30S subunit and EF-Tu at the factor-binding site. A series of conformational changes in EF-Tu and aminoacyl-tRNA suggest a communication pathway between the decoding center and the GTPase center of EF-Tu.
PMCID: PMC3763470  PMID: 19833920
EF-Tu; 70S; ribosome; decoding; translation; aminoacyl-tRNA; tRNA delivery; protein synthesis; A/T; GTP; kirromycin
4.  YoeB–ribosome structure: a canonical RNase that requires the ribosome for its specific activity 
Nucleic Acids Research  2013;41(20):9549-9556.
As a typical endoribonuclease, YoeB mediates cellular adaptation in diverse bacteria by degrading mRNAs on its activation. Although the catalytic core of YoeB is thought to be identical to well-studied nucleases, this enzyme specifically targets mRNA substrates that are associated with ribosomes in vivo. However, the molecular mechanism of mRNA recognition and cleavage by YoeB, and the requirement of ribosome for its optimal activity, largely remain elusive. Here, we report the structure of YoeB bound to 70S ribosome in pre-cleavage state, revealing that both the 30S and 50S subunits participate in YoeB binding. The mRNA is recognized by the catalytic core of YoeB, of which the general base/acid (Glu46/His83) are within hydrogen-bonding distance to their reaction atoms, demonstrating an active conformation of YoeB on ribosome. Also, the mRNA orientation involves the universally conserved A1493 and G530 of 16S rRNA. In addition, mass spectrometry data indicated that YoeB cleaves mRNA following the second position at the A-site codon, resulting in a final product with a 3′–phosphate at the newly formed 3′ end. Our results demonstrate a classical acid-base catalysis for YoeB-mediated RNA hydrolysis and provide insight into how the ribosome is essential for its specific activity.
PMCID: PMC3814384  PMID: 23945936
5.  Crystal Structure of 70S Ribosome with Both Cognate tRNAs in the E and P Sites Representing an Authentic Elongation Complex 
PLoS ONE  2013;8(3):e58829.
During the translation cycle, a cognate deacylated tRNA can only move together with the codon into the E site. We here present the first structure of a cognate tRNA bound to the ribosomal E site resulting from translocation by EF-G, in which an entire L1 stalk (L1 protein and L1 rRNA) interacts with E-site tRNA (E-tRNA), representing an authentic ribosome elongation complex. Our results revealed that the Watson-Crick base pairing is formed at the first and second codon-anticodon positions in the E site in the ribosome elongation complex, whereas the codon-anticodon interaction in the third position is indirect. Analysis of the observed conformations of mRNA and E-tRNA suggests that the ribosome intrinsically has the potential to form codon-anticodon interaction in the E site, independently of the mRNA configuration. We also present a detailed description of the biologically relevant position of the entire L1 stalk and its interacting cognate E-tRNA, which provides a better understanding of the structural basis for translation elongation. Furthermore, to gain insight into translocation, we report the positioning of protein L6 contacting EF-G, as well as the conformational change of the C-terminal tail of protein S13 in the decoding center.
PMCID: PMC3602588  PMID: 23527033
6.  Ribosome engineering to promote new crystal forms 
Truncation of ribosomal protein L9 in T. thermophilus allows the generation of new crystal forms and the crystallization of ribosome–GTPase complexes.
Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-­G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.
PMCID: PMC3335287  PMID: 22525755
ribosome; GTPase; engineering
7.  The Structural Basis for mRNA Recognition and Cleavage by the Ribosome-Dependent Endonuclease RelE 
Cell  2009;139(6):1084-1095.
Translational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 Å) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 Å) and after (3.6 Å) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2′-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage.
PMCID: PMC2807027  PMID: 20005802
8.  The structure of Pyrococcus horikoshii 2′-5′ RNA ligase at 1.94 Å resolution reveals a possible open form with a wider active-site cleft 
The crystal structure of the 2′-5′ RNA ligase PH0099 from P. horikoshii OT3 was solved at 1.94 Å resolution. The molecule has a bilobal α+β arrangement with two antiparallel β-sheets constituting a V-shaped active-site cleft, as found in other members of the 2H phosphoesterase superfamily.
Bacterial and archaeal 2′-5′ RNA ligases, members of the 2H phosphoesterase superfamily, catalyze the linkage of the 5′ and 3′ exons via a 2′-5′-phosphodiester bond during tRNA-precursor splicing. The crystal structure of the 2′-5′ RNA ligase PH0099 from Pyrococcus horikoshii OT3 was solved at 1.94 Å resolution (PDB code 1vgj). The molecule has a bilobal α+β arrangement with two antiparallel β-sheets constituting a V-shaped active-site cleft, as found in other members of the 2H phosphoesterase superfamily. The present structure was significantly different from that determined previously at 2.4 Å resolution (PDB code 1vdx) in the active-site cleft; the entrance to the cleft is wider and the active site is easily accessible to the substrate (RNA precursor) in our structure. Structural comparison with the 2′-5′ RNA ligase from Thermus thermophilus HB8 also revealed differences in the RNA precursor-binding region. The structural differences in the active-site residues (tetrapeptide motifs H-X-T/S-X) between the members of the 2H phosphoesterase superfamily are discussed.
PMCID: PMC2225383  PMID: 17142895
2′-5′ RNA ligase; 2H phosphoesterase superfamily; Pyrococcus horikoshii
9.  Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in l-lactate and sugar utilization 
Nucleic Acids Research  2008;36(22):7110-7123.
LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-Å resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn2+ binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed.
PMCID: PMC2602784  PMID: 18988622
10.  Preparation, crystallization and preliminary X-ray diffraction analysis of PH1948, predicted RNA methyltransferase from Pyrococcus horikoshii *  
RNA methyltransferase is responsible for transferring methyl and resulting in methylation on the bases or ribose ring of RNA, which existed widely but mostly remains an open question. A recombinant protein PH1948 predicting RNA methyltransferase from Pyrococcus horikoshii OT3 has been crystallized. The crystals of selenomethionyl PH1948 belong to space group C2, with unit-cell parameters a=207.0 Å, b=43.1 Å, c=118.2 Å, β=92.1°, and diffract X-rays to 2.2 Å resolution. The V M value was determined to be 2.8 Å3/Da, indicating the presence of four protein molecules in the asymmetric unit.
PMCID: PMC1389872  PMID: 15909326
Pyrococcus horikoshii; Methyltransferase; X-ray diffraction; Protein crystallography

Results 1-10 (10)