Shun-ichi Sekine

Structural Basis for Transcription Regulation by Alarmone ppGpp

Guanosine-tetraphosphate (ppGpp) is a major regulator of stringent control, an adaptive response of bacteria to amino acid starvation. The 2.7 A resolution structure of the Thermus thermophilus RNA polymerase (RNAP) holoenzyme in complex with ppGpp reveals that ppGpp binds to the same site near the active center in both independent RNAP molecules in the crystal but in strikingly distinct orientations. Binding is symmetrical with respect to the two diphosphates of ppGpp and is relaxed with respect to the orientation of the nucleotide base. Different modes of ppGpp binding are coupled with asymmetry of the active site configurations. The results suggest that base pairing of ppGpp with cytosines in the nontemplate DNA strand might be an essential component of transcription control by ppGpp. We present experimental evidence highlighting the importance of base-specific contacts between ppGpp and specific cytosine residues during both transcription initiation and elongation.

Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein

The multi-subunit DNA-dependent RNA polymerase (RNAP) is the principal enzyme of transcription for gene expression. Transcription is regulated by various transcription factors. Gre factor homologue 1 (Gfh1), found in the Thermus genus, is a close homologue of the well-conserved bacterial transcription factor GreA, and inhibits transcription initiation and elongation by binding directly to RNAP. The structural basis of transcription inhibition by Gfh1 has remained elusive, although the crystal structures of RNAP and Gfh1 have been determined separately. Here we report the crystal structure of Thermus thermophilus RNAP complexed with Gfh1. The amino-terminal coiled-coil domain of Gfh1 fully occludes the channel formed between the two central modules of RNAP; this channel would normally be used for nucleotide triphosphate (NTP) entry into the catalytic site. Furthermore, the tip of the coiled-coil domain occupies the NTP β-γ phosphate-binding site. The NTP-entry channel is expanded, because the central modules are ‘ratcheted’ relative to each other by ∼7°, as compared with the previously reported elongation complexes. This ‘ratcheted state’ is an alternative structural state, defined by a newly acquired contact between the central modules. Therefore, the shape of Gfh1 is appropriate to maintain RNAP in the ratcheted state. Simultaneously, the ratcheting expands the nucleic-acid-binding channel, and kinks the bridge helix, which connects the central modules. Taken together, the present results reveal that Gfh1 inhibits transcription by preventing NTP binding and freezing RNAP in the alternative structural state. The ratcheted state might also be associated with other aspects of transcription, such as RNAP translocation and transcription termination.

ATP binding by glutamyl‐tRNA synthetase is switched to the productive mode by tRNA binding

Aminoacyl‐tRNA synthetases catalyze the formation of an aminoacyl‐AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA. A subset of aminoacyl‐tRNA synthetases, including glutamyl‐tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl‐AMP formation in the absence of tRNA. In this study, we determined the crystal structure of the ‘non‐productive’ complex of Thermus thermophilus GluRS, ATP and L‐glutamate, together with those of the GluRS·ATP, GluRS·tRNA·ATP and GluRS·tRNA·GoA (a glutamyl‐AMP analog) complexes. In the absence of tRNAGlu, ATP is accommodated in a ‘non‐productive’ subsite within the ATP‐binding site, so that the ATP α‐phosphate and the glutamate α‐carboxyl groups in GluRS· ATP·Glu are too far from each other (6.2 Å) to react. In contrast, the ATP‐binding mode in GluRS·tRNA· ATP is dramatically different from those in GluRS·ATP·Glu and GluRS·ATP, but corresponds to the AMP moiety binding mode in GluRS·tRNA·GoA (the ‘productive’ subsite). Therefore, tRNA binding to GluRS switches the ATP‐binding mode. The interactions of the three tRNAGlu regions with GluRS cause conformational changes around the ATP‐binding site, and allow ATP to bind to the ‘productive’ subsite.

Structural basis for functional mimicry of long-variable-arm tRNA by transfer-messenger RNA

tmRNA and small protein B (SmpB) are essential trans-translation system components. In the present study, we determined the crystal structure of SmpB in complex with the entire tRNA domain of the tmRNA from Thermus thermophilus. Overall, the ribonucleoprotein complex (tRNP) mimics a long-variable-arm tRNA (class II tRNA) in the canonical L-shaped tertiary structure. The tmRNA terminus corresponds to the acceptor and T arms, or the upper part, of tRNA. On the other hand, the SmpB protein simulates the lower part, the anticodon and D stems, of tRNA. Intriguingly, several amino acid residues collaborate with tmRNA bases to reproduce the canonical tRNA core layers. The linker helix of tmRNA had been considered to correspond to the anticodon stem, but the complex structure unambiguously shows that it corresponds to the tRNA variable arm. The tmRNA linker helix, as well as the long variable arm of class II tRNA, may occupy the gap between the large and small ribosomal subunits. This suggested how the tRNA domain is connected to the mRNA domain entering the mRNA channel. A loop of SmpB in the tRNP is likely to participate in the interaction with alanyl-tRNA synthetase, which may be the mechanism for the promotion of tmRNA alanylation by the SmpB protein. Therefore, the tRNP may simulate a tRNA, both structurally and functionally, with respect to aminoacylation and ribosome entry.

The selective tRNA aminoacylation mechanism based on a single G•U pair

Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNAAla selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3•U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNAAla with G3•U70 and its A3•U70 variant. AlaRS interacts with both the minor- and the major-groove sides of G3•U70, widening the major groove. The geometry difference between G3•U70 and A3•U70 is transmitted along the acceptor stem to the 3′-CCA region. Thus, the 3′-CCA region of tRNAAla with G3•U70 is oriented to the reactive route that reaches the active site, whereas that of the A3•U70 variant is folded back into the non-reactive route. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat.

Crystal Structure of tRNA Adenosine Deaminase (TadA) from Aquifex aeolicus

The bacterial tRNA adenosine deaminase (TadA) generates inosine by deaminating the adenosine residue at the wobble position of tRNAArg-2. This modification is essential for the decoding system. In this study, we determined the crystal structure of Aquifex aeolicus TadA at a 1.8-Å resolution. This is the first structure of a deaminase acting on tRNA. A. aeolicus TadA has an α/β/α three-layered fold and forms a homodimer. The A. aeolicus TadA dimeric structure is completely different from the tetrameric structure of yeast CDD1, which deaminates mRNA and cytidine, but is similar to the dimeric structure of yeast cytosine deaminase. However, in the A. aeolicus TadA structure, the shapes of the C-terminal helix and the regions between the β4 and β5 strands are quite distinct from those of yeast cytosine deaminase and a large cavity is produced. This cavity contains many conserved amino acid residues that are likely to be involved in either catalysis or tRNA binding. We made a docking model of TadA with the tRNA anticodon stem loop.

Structural basis for mutual relief of the Rac guanine nucleotide exchange factor DOCK2 and its partner ELMO1 from their autoinhibited forms

DOCK2, a hematopoietic cell-specific, atypical guanine nucleotide exchange factor, controls lymphocyte migration through ras-related C3 botulinum toxin substrate (Rac) activation. Dedicator of cytokinesis 2–engulfment and cell motility protein 1 (DOCK2•ELMO1) complex formation is required for DOCK2-mediated Rac signaling. In this study, we identified the N-terminal 177-residue fragment and the C-terminal 196-residue fragment of human DOCK2 and ELMO1, respectively, as the mutual binding regions, and solved the crystal structure of their complex at 2.1-Å resolution. The C-terminal Pro-rich tail of ELMO1 winds around the Src-homology 3 domain of DOCK2, and an intermolecular five-helix bundle is formed. Overall, the entire regions of both DOCK2 and ELMO1 assemble to create a rigid structure, which is required for the DOCK2•ELMO1 binding, as revealed by mutagenesis. Intriguingly, the DOCK2•ELMO1 interface hydrophobically buries a residue which, when mutated, reportedly relieves DOCK180 from autoinhibition. We demonstrated that the ELMO-interacting region and the DOCK-homology region 2 guanine nucleotide exchange factor domain of DOCK2 associate with each other for the autoinhibition, and that the assembly with ELMO1 weakens the interaction, relieving DOCK2 from the autoinhibition. The interactions between the N- and C-terminal regions of ELMO1 reportedly cause its autoinhibition, and binding with a DOCK protein relieves the autoinhibition for ras homolog gene family, member G binding and membrane localization. In fact, the DOCK2•ELMO1 interface also buries the ELMO1 residues required for the autoinhibition within the hydrophobic core of the helix bundle. Therefore, the present complex structure reveals the structural basis by which DOCK2 and ELMO1 mutually relieve their autoinhibition for the activation of Rac1 for lymphocyte chemotaxis.

Decameric SelA•tRNASec Ring Structure Reveals Mechanism of Bacterial Selenocysteine Formation

Putting Selenium in Proteins The 21st amino acid, selenocysteine (Sec), occurs in the active site of many redox enzymes. Its cognate transfer RNA (tRNA) is first loaded with Ser by seryl-tRNA synthetase and the Ser-tRNASec is then converted to Sec-tRNASec. Itoh et al. (p. 75) determined the crystal structures of the selenocysteine synthase, SelA, that is responsible for this conversion in bacteria, alone and in complex with tRNA. The decameric SelA complex binds to 10 tRNASec molecules. The structures, together with biochemistry, show how SelA discriminates tRNASec from tRNASer, give insight into the mechanism of catalysis, and show that decamerization is essential for function. Structural and biochemical data reveal how selenocysteine is produced from serine on transfer RNA. The 21st amino acid, selenocysteine (Sec), is synthesized on its cognate transfer RNA (tRNASec). In bacteria, SelA synthesizes Sec from Ser-tRNASec, whereas in archaea and eukaryotes SepSecS forms Sec from phosphoserine (Sep) acylated to tRNASec. We determined the crystal structures of Aquifex aeolicus SelA complexes, which revealed a ring-shaped homodecamer that binds 10 tRNASec molecules, each interacting with four SelA subunits. The SelA N-terminal domain binds the tRNASec-specific D-arm structure, thereby discriminating Ser-tRNASec from Ser-tRNASer. A large cleft is created between two subunits and accommodates the 3′-terminal region of Ser-tRNASec. The SelA structures together with in vivo and in vitro enzyme assays show decamerization to be essential for SelA function. SelA catalyzes pyridoxal 5′-phosphate–dependent Sec formation involving Arg residues nonhomologous to those in SepSecS. Different protein architecture and substrate coordination of the bacterial enzyme provide structural evidence for independent evolution of the two Sec synthesis systems present in nature.

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

Alanyl-tRNA synthetase (AlaRS) specifically recognizes the major identity determinant, the G3:U70 base pair, in the acceptor stem of tRNAAla by both the tRNA-recognition and editing domains. In this study, we solved the crystal structures of 2 halves of Archaeoglobus fulgidus AlaRS: AlaRS-ΔC, comprising the aminoacylation, tRNA-recognition, and editing domains, and AlaRS-C, comprising the dimerization domain. The aminoacylation/tRNA-recognition domains contain an insertion incompatible with the class-specific tRNA-binding mode. The editing domain is fixed tightly via hydrophobic interactions to the aminoacylation/tRNA-recognition domains, on the side opposite from that in threonyl-tRNA synthetase. A groove formed between the aminoacylation/tRNA-recognition domains and the editing domain appears to be an alternative tRNA-binding site, which might be used for the aminoacylation and/or editing reactions. Actually, the amino acid residues required for the G3:U70 recognition are mapped in this groove. The dimerization domain consists of helical and globular subdomains. The helical subdomain mediates dimerization by forming a helix–loop–helix zipper. The globular subdomain, which is important for the aminoacylation and editing activities, has a positively-charged face suitable for tRNA binding.

Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase

To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the “editing” mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the β subunit. We report the crystal structure of an N-terminal fragment of the PheRS β subunit (PheRS-βN) from the archaeon, Pyrococcus horikoshii, at 1.94-Å resolution. PheRS-βN includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea/eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3/4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe, indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3′-terminal adenosine, reduced Tyr-tRNAPhe deacylation activity. Third, the mutations of archaea/eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNAPhe deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis.