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1.  Structural basis of transcriptional pausing in bacteria 
Cell  2013;152(3):431-441.
Transcriptional pausing by multi-subunit RNA Polymerases (RNAPs) is a key mechanism for regulating gene expression in both prokaryotes and eukaryotes, and is a prerequisite for transcription termination. Pausing and termination states are thought to arise through a common, elemental pause state that is inhibitory for nucleotide addition. We report three crystal structures of Thermus RNAP elemental paused elongation complexes (ePECs). The structures reveal the same relaxed, open-clamp RNAP conformation in the ePEC that may arise by failure to reestablish DNA contacts during translocation. A kinked bridge-helix sterically blocks the RNAP active site, explaining how this conformation inhibits RNAP catalytic activity. Our results provide a framework for understanding how RNA hairpin formation stabilizes the paused state and how the ePEC intermediate facilitates termination.
PMCID: PMC3564060  PMID: 23374340
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.  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
4.  Mechanism of expanding the decoding capacity of tRNAs by modification of uridines 
One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNAVal containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We see unusual base pairs with the three non-complementary codon bases, including a GU base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.
PMCID: PMC2816034  PMID: 17496902
5.  Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome 
Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. Additionally, though structures of the 50S subunit found no ordered proteins at the PTC, biochemical evidence suggests specific proteins are capable of interacting with the 3′ ends of the tRNA ligands. Here we present structures at 3.5 Å and 3.55 Å resolution of the 70S ribosome in complex with A- and P-site tRNAs that mimic pre- and post-peptidyl transfer states. These structures demonstrate that the PTC is very similar between the 50S subunit and the intact ribosome. Additionally they reveal interactions between ribosomal proteins L16 and L27 and the tRNA substrates, helping to elucidate the role of these proteins in peptidyl transfer.
PMCID: PMC2679717  PMID: 19363482
6.  Insights into translational termination from the structure of RF2 bound to the ribosome 
Science (New York, N.Y.)  2008;322(5903):953-956.
The termination of protein synthesis occurs through specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site tRNA. Here we present the crystal structure at 3.5 Å resolution of release factor RF2 in complex with its cognate UGA stop codon in the 70S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon and suggests a model for the role of a universally conserved GGQ motif in catalysis of peptide release.
PMCID: PMC2642913  PMID: 18988853
7.  Determination of thermodynamic parameters for HIV DIS type loop–loop kissing complexes 
Nucleic Acids Research  2004;32(17):5126-5133.
The HIV-1 type dimerization initiation signal (DIS) loop was used as a starting point for the analysis of the stability of Watson–Crick (WC) base pairs in a tertiary structure context. We used ultraviolet melting to determine thermodynamic parameters for loop–loop tertiary interactions and compared them with regular secondary structure RNA helices of the same sequences. In 1 M Na+ the loop–loop interaction of a HIV-1 DIS type pairing is 4 kcal/mol more stable than its sequence in an equivalent regular and isolated RNA helix. This difference is constant and sequence independent, suggesting that the rules governing the stability of WC base pairs in the secondary structure context are also valid for WC base pairs in the tertiary structure context. Moreover, the effect of ion concentration on the stability of loop–loop tertiary interactions differs considerably from that of regular RNA helices. The stabilization by Na+ and Mg2+ is significantly greater if the base pairing occurs within the context of a loop–loop interaction. The dependence of the structural stability on salt concentration was defined via the slope of a Tm/log [ion] plot. The short base-paired helices are stabilized by 8°C/log [Mg2+] or 11°C/log [Na+], whereas base-paired helices forming tertiary loop–loop interactions are stabilized by 16°C/log [Mg2+] and 26°C/log [Na+]. The different dependence on ionic strength that is observed might reflect the contribution of specific divalent ion binding to the preformation of the hairpin loops poised for the tertiary kissing loop–loop contacts.
PMCID: PMC521654  PMID: 15459283

Results 1-7 (7)