The Giardia lamblia ribosome structure reveals divergence in several biological pathways and the mode of emetine function.

Giardia lamblia is a deeply branching protist and a human pathogen. Its unusual biology presents the opportunity to explore conserved and fundamental molecular mechanisms. We determined the structure of the G. lamblia 80S ribosome bound to tRNA, mRNA, and the antibiotic emetine by cryo-electron microscopy, to an overall resolution of 2.49 Å. The structure reveals rapidly evolving protein and nucleotide regions, differences in the peptide exit tunnel, and likely altered ribosome quality control pathways. Examination of translation initiation factor binding sites suggests these interactions are conserved despite a divergent initiation mechanism. Highlighting the potential of G. lamblia to resolve conserved biological principles; our structure reveals the interactions of the translation inhibitor emetine with the ribosome and mRNA, thus providing insight into the mechanism of action for this widely used antibiotic. Our work defines key questions in G. lamblia and motivates future experiments to explore the diversity of eukaryotic gene regulation.

Unifying the Aminohexopyranose- and Peptidyl-Nucleoside Antibiotics: Implications for Antibiotic Design.

In search of new anti-tuberculars compatible with anti-retroviral therapy we re-identified amicetin as a lead compound. Amicetin's binding to the 70S ribosomal subunit of Thermus thermophilus (Tth) has been unambiguously determined by crystallography and reveals it to occupy the peptidyl transferase center P-site of the ribosome. The amicetin binding site overlaps significantly with that of the well-known protein synthesis inhibitor balsticidin S. Amicetin, however, is the first compound structurally characterized to bind to the P-site with demonstrated selectivity for the inhibition of prokaryotic translation. The natural product-ribosome structure enabled the synthesis of simplified analogues that retained both potency and selectivity for the inhibition of prokaryotic translation.

Computational design of three-dimensional RNA structure and function.

RNA nanotechnology seeks to create nanoscale machines by repurposing natural RNA modules. The field is slowed by the current need for human intuition during three-dimensional structural design. Here, we demonstrate that three distinct problems in RNA nanotechnology can be reduced to a pathfinding problem and automatically solved through an algorithm called RNAMake. First, RNAMake discovers highly stable single-chain solutions to the classic problem of aligning a tetraloop and its sequence-distal receptor, with experimental validation from chemical mapping, gel electrophoresis, solution X-ray scattering and crystallography with 2.55 Å resolution. Second, RNAMake automatically generates structured tethers that integrate 16S and 23S ribosomal RNAs into single-chain ribosomal RNAs that remain uncleaved by ribonucleases and assemble onto messenger RNA. Third, RNAMake enables the automated stabilization of small-molecule binding RNAs, with designed tertiary contacts that improve the binding affinity of the ATP aptamer and improve the fluorescence and stability of the Spinach RNA in cell extracts and in living Escherichia coli cells.

Structured RNAs that evade or confound exonucleases: function follows form.

Cells contain powerful RNA decay machinery to eliminate unneeded RNA from the cell, and this process is an important and regulated part of controlling gene expression. However, certain structured RNAs have been found that can robustly resist degradation and extend the lifetime of an RNA. In this review, we present three RNA structures that use a specific three-dimensional fold to provide protection from RNA degradation, and discuss how the recently-solved structures of these RNAs explain their function. Specifically, we describe the Xrn1-resistant RNAs from arthropod-borne flaviviruses, exosome-resistant long non-coding RNAs associated with lung cancer metastasis and found in Kaposi's sarcoma-associated herpesvirus, and tRNA-like sequences occurring in certain plant viruses. These three structures reveal three different mechanisms to protect RNAs from decay and suggest RNA structure-based nuclease resistance may be a widespread mechanism of regulation.

Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog.

Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme.

Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-Å resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5' to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-Å resolution exhibits a disordered scissile phosphate and nucleotide 5' to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2'-hydroxyl of the nucleotide 5' to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket.

Initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5B and mechanism among translational GTPases.

The initiation of protein synthesis uses initiation factor 2 (IF2) in prokaryotes and a related protein named eukaryotic initiation factor 5B (eIF5B) in eukaryotes. IF2 is a GTPase that positions the initiator tRNA on the 30S ribosomal initiation complex and stimulates its assembly to the 50S ribosomal subunit to make the 70S ribosome. The 3.1-Å resolution X-ray crystal structures of the full-length Thermus thermophilus apo IF2 and its complex with GDP presented here exhibit two different conformations (all of its domains except C2 domain are visible). Unlike all other translational GTPases, IF2 does not have an effecter domain that stably contacts the switch II region of the GTPase domain. The domain organization of IF2 is inconsistent with the "articulated lever" mechanism of communication between the GTPase and initiator tRNA binding domains that has been proposed for eIF5B. Previous cryo-electron microscopy reconstructions, NMR experiments, and this structure show that IF2 transitions from being flexible in solution to an extended conformation when interacting with ribosomal complexes.

Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor.

Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl(fMet)-tRNA(fMet) positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process.

tRNAHis-guanylyltransferase establishes tRNAHis identity.

Histidine transfer RNA (tRNA) is unique among tRNA species as it carries an additional nucleotide at its 5' terminus. This unusual G(-1) residue is the major tRNA(His) identity element, and essential for recognition by the cognate histidyl-tRNA synthetase to allow efficient His-tRNA(His) formation. In many organisms G(-1) is added post-transcriptionally as part of the tRNA maturation process. tRNA(His) guanylyltransferase (Thg1) specifically adds the guanylyate residue by recognizing the tRNA(His) anticodon. Thg1 homologs from all three domains of life have been the subject of exciting research that gave rise to a detailed biochemical, structural and phylogenetic enzyme characterization. Thg1 homologs are phylogenetically classified into eukaryal- and archaeal-type enzymes differing characteristically in their cofactor requirements and specificity. Yeast Thg1 displays a unique but limited ability to add 2-3 G or C residues to mutant tRNA substrates, thus catalyzing a 3' → 5' RNA polymerization. Archaeal-type Thg1, which has been horizontally transferred to certain bacteria and few eukarya, displays a more relaxed substrate range and may play additional roles in tRNA editing and repair. The crystal structure of human Thg1 revealed a fascinating structural similarity to 5' → 3' polymerases, indicating that Thg1 derives from classical polymerases and evolved to assume its specific function in tRNA(His) processing.

Programing the formation of DNA and PNA quadruplexes by pi-pi-stacking interactions.

Guanine (G) rich G(4)T(4)G(4) DNA and homologous PNA strands tend to form antiparallel dimeric quadruplexes. In contrast, the same DNA strands carrying planar aromatic 5'-residues preferentially form parallel DNA quadruplex. Conformation and composition of the DNA quadruplexes can be programed by pi-pi-stacking interaction exerted by the 5'-residues.

Energetic rationale for an unexpected and abrupt reversal of guanidinium chloride-induced unfolding of peptide deformylase.

Peptide deformylase (PDF) catalyzes the removal of formyl group from the N-terminal methionine residues of nascent proteins in prokaryotes, and this enzyme is a high priority target for antibiotic design. In pursuit of delineating the structural-functional features of Escherichia coli PDF (EcPDF), we investigated the mechanistic pathway for the guanidinium chloride (GdmCl)-induced unfolding of the enzyme by monitoring the secondary structural changes via CD spectroscopy. The experimental data revealed that EcPDF is a highly stable enzyme, and it undergoes slow denaturation in the presence of varying concentrations of GdmCl. The most interesting aspect of these studies has been the abrupt reversal of the unfolding pathway at low to moderate concentrations of the denaturant, but not at high concentration. An energetic rationale for such an unprecedented feature in protein chemistry is offered.

Spacer-based selectivity in the binding of "two-prong" ligands to recombinant human carbonic anhydrase I.

Benzenesulfonamide and iminodiacetate (IDA)-conjugated Cu(2+) independently interact at the active site and a peripheral site of carbonic anhydrases, respectively [Banerjee, A. L., Swanson, M., Roy, B. C., Jia, X., Haldar, M. K., Mallik, S., and Srivastava, D. K. (2004) J. Am. Chem. Soc. 126, 10875-10883]. By attaching IDA-bound Cu(2+) to benzenesulfonamide via different chain length spacers, we synthesized two "two-prong" ligands, L1 and L2, in which the distances between Cu(2+) and NH(2) group of sulfonamide were 29 and 22 A, respectively. We compared the binding affinities of L1 and L2, vis-a-vis their parent compound, benzenesulfonamide, for recombinant human carbonic anhydrase I (hCA-I) by performing the fluorescence titration and steady-state kinetic experiments. The experimental data revealed that whereas the binding affinity of L1 for hCA-I was similar to that of benzenesulfonamide, the binding affinity of L2 was approximately 2 orders of magnitude higher, making L2 one of the most potent ligands or inhibitors of hCA-I. Since the enhanced binding or inhibitory potency of L2 is diminished (to the level of benzenesulfonamide) either in the presence of EDTA or upon treatment of the enzyme with diethyl pyrocarbonate, it is proposed that Cu(2+) of L2 interacts with one of the surface-exposed histidine residues of the enzyme. A cumulative account of the experimental data leads to the suggestion that the differential binding of L1 versus L2 to hCA-I is encoded in the chain length of the spacer moiety.