Structure of an archaeal homolog of the eukaryotic RNA polymerase II RPB4/RPB7 complex.

The eukaryotic subunits RPB4 and RPB7 form a heterodimer that reversibly associates with the RNA polymerase II core and constitute the only two components of the enzyme for which no structural information is available. We have determined the crystal structure of the complex between the Methanococcus jannaschii subunits E and F, the archaeal homologs of RPB7 and RPB4. Subunit E has an elongated two-domain structure and contains two potential RNA binding motifs, while the smaller F subunit wraps around one side of subunit E, at the interface between the two domains. We propose a model for the interaction between RPB4/RPB7 and the core RNA polymerase in which the RNA binding face of RPB7 is positioned to interact with the nascent RNA transcript.

Electrical bone graft stimulation for spinal fusion: a review.

Although electrical stimulation to aid bone fusion is well established in the treatment of long-bone fractures, its use as an aid in spinal fusion is not as well documented. This article presents the history and scientific basis of electrical stimulation to aid bone fusion and extensively reviews the clinical literature. It is intended to provide an objective review of the indications and limitations of electrical stimulation to enhance spinal fusion and to serve as a reference source for further study.

Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding.

Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding.

Active site of lysyl-tRNA synthetase: structural studies of the adenylation reaction.

Aminoacyl-tRNA synthetases play a key role in protein biosynthesis by catalyzing the specific aminoacylation of tRNA. The energy required for the formation of the ester bond between the amino acid carboxylate group and the tRNA acceptor stem is supplied by coupling the reaction to the hydrolysis of ATP. Lysyl-tRNA synthetase from Escherichia coli belongs to the family of class II synthetases and carries out a two-step reaction, in which lysine is activated by being attached to the alpha-phosphate of AMP before being transferred to the cognate tRNA. Crystals of the thermo-inducible E. coli lysyl-tRNA synthetase LysU which diffract to 2.1 A resolution have been used to determine crystal structures of the enzyme in the presence of lysine, the lysyl-adenylate intermediate, and the nonhydrolyzable ATP analogue AMP-PCP. Additional data have been obtained from crystals soaked in a solution containing ATP and Mn(2+). The refined crystal structures give "snapshots" of the active site corresponding to key steps in the aminoacylation reaction and provide the structural framework for understanding the mechanism of lysine activation. The active site of LysU is shaped to position the substrates for the nucleophilic attack of the lysine carboxylate on the ATP alpha-phosphate. No residues are directly involved in catalysis, but a number of highly conserved amino acids and three metal ions coordinate the substrates and stabilize the pentavalent transition state. A loop close to the catalytic pocket, disordered in the lysine-bound structure, becomes ordered upon adenine binding.

Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and transcription factor interaction target.

Eukaryotic nuclei contain three different types of RNA polymerases (RNAPs), each consisting of 12-18 different subunits. The evolutionarily highly conserved RNAP subunit RPB5 is shared by all three enzymes and therefore represents a key structural/functional component of all eukaryotic RNAPs. Here we present the crystal structure of the RPB5 subunit from Saccharomyces cerevisiae. The bipartite structure includes a eukaryote-specific N-terminal domain and a C-terminal domain resembling the archaeal RNAP subunit H. RPB5 has been implicated in direct protein-protein contacts with transcription factor IIB, one of the components of the RNAP(II) basal transcriptional machinery, and gene-specific activator proteins, such as the hepatitis B virus transactivator protein X. The experimentally mapped regions of RPB5 involved in these interactions correspond to distinct and surface-exposed alpha-helical structures.

Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii.

We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase alpha-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg(2+), Mn(2+)or Zn(2+), and is additionally stimulated by the monovalent cation K(+). A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have been shown to be essential for catalysis in the mouse primase. Of the four cysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first report on the biochemical characterisation of an archaeal DNA primase.

Crystallization and preliminary diffraction studies of the RNA polymerase subunit RPB5 from Saccharomyces cerevisiae.

Crystals of the RNA polymerase subunit RPB5 from Saccharomyces cerevisiae have been obtained by vapour-diffusion techniques. The protein has been overexpressed in bacterial cells as a fusion with glutathione S-transferase. Two monoclinic crystal forms can be grown under different sets of conditions. In both cases, the diffraction is consistent with space group P21, with unit-cell parameters a = 45. 3, b = 135.3, c = 47.3 A, beta = 118.6 degrees for crystal form I and a = 48.4, b = 137.1, c = 47.1 A, beta = 118.6 degrees for crystal form II.

Structure of the Kunitz-type soybean trypsin inhibitor (STI): implication for the interactions between members of the STI family and tissue-plasminogen activator.

The Kunitz-type soybean trypsin inhibitor (STI) has played a key role in the early study of proteinases, having been used as the main substrate in the biochemical and kinetic work that led to the definition of the standard mechanism of action of proteinase inhibitors. A partial structure of STI complexed with porcine trypsin has previously been reported, in which the first 93 residues of the inhibitor, including the region of contact with trypsin, were relatively well defined, whereas for the remaining part of the peptide chain only some Calpha atoms were located. The structure of the inhibitor in its free form has now been determined by molecular replacement to 2.5 A, using the coordinates of the homologous Erythrina trypsin inhibitor as a search model. When the refined atomic coordinates of STI are compared with the partial model previously available, the conformation of the reactive-site loop and its position with respect to the main body of the molecule does not change when the inhibitor interacts with trypsin. There are instead, despite the high similarity in the overall tertiary structure, significant differences between STI and Erythrina trypsin inhibitor (ETI) in the region which is in contact with the enzyme in the STI:trypsin crystal structure. Some of these differences can explain the unique specificity of ETI and its ability to inhibit the fibrinolytic enzyme tissue-type plasminogen activator.

Transparaspinal exposure of dumbbell tumors of the spine. Report of two cases.

The authors present a surgical technique for resection of dumbbell tumors of the spine. The transparaspinal exposure combines laminectomy and sectioning of the paraspinal muscles through a transverse incision. The procedure allows total tumor resection by means of a single posterior approach in selected patients, thus obviating the need for a combined anteroposterior operation. The advantages and disadvantages of the transparaspinal approach compared with the more extensive lateral extracavitary approach are discussed.

Paraganglioma of the filum terminale: case report and literature review.

A rare case of a paraganglioma of the filum terminale is reported. The patient presented with headache and papilledema. Magnetic resonance imaging demonstrated enlarged ventricles of the brain and a well-demarcated intradural spine tumor. After resection of the tumor, the papilledema and headaches resolved, and the ventricles returned to normal size. Pathologic examination revealed a paraganglioma of the filum terminale. We present this rare case and review the literature of spinal paragangliomas as well as the association of spinal tumors with hydrocephalus.

[Surgical approach to benign extradural lesions of the thoracic spine].

A benign epidural lesion in the thoracic spine is rare, and usually the result of intervertebral disc herniation or infection. Not long ago patients were diagnosed late in the course of their disease and the surgical results of the standard laminectomy usually performed were grave. The development of newer imaging techniques (CT and MRI) has made diagnosis much easier, so diagnosis is often earlier, when neurological deficit is minimal. Newer neurosurgical techniques and approaches to the thoracic spoine have been developed to treat these lesions, which we describe. Clinical data on 16 patients operated from January 1996 to January 1997 are presented.

Dorsal endodermal cyst of the upper cervical spine.

This report describes a case of a rare, dorsally placed enterogenous cyst at the craniocervical junction. The patient had preoperative magnetic resonance imaging studies, followed by microsurgical removal of the cyst. The patient made an uneventful recovery from the surgery. Pathological examination revealed an enterogenous cyst. Although enterogenous cysts are more commonly found in the lower cervical or thoracic spine, it is important to recognize that they may also be found at the craniocervical junction. In addition, cysts may occur posterior to the chord. Microsurgical removal is usually effective in the treatment of these cysts.

False localizing signs in upper cervical spinal cord compression.

Proprioceptive loss, paresthesias, and atrophy of the hands can occur with disorders afflicting the upper cervical spinal cord. The diagnosis might be erroneous, because compression in this region might produce signs and symptoms that seem to originate in the lower cervical cord. This article reviews the clinical presentation and radiographic data of a consecutive series of 11 patients who presented between 1992 and 1994 with an extradural lesion above the C4 level. Each patient had a characteristic syndrome of finger and hand dysesthesia, hand atrophy, and occipital or cervical pain. These complaints usually preceded the development of spasticity and gait disturbance. Initial diagnoses included brachial plexopathy, shoulder dysfunction, viral syndrome, and cervical spondylosis at a lower segment. Cervical spondylosis or a herniated disc was the most common pathogenesis. The most commonly involved level was C3-C4. Nine patients underwent a surgical procedure; eight showed significant postoperative improvement (mean time of follow-up examination, 9.7 mo; follow-up range, 1-24 mo). One patient was lost to follow-up. Although the pathophysiology of these findings is unknown, theories include anterior spinal artery ischemia, venous obstruction, and differential decussation of the forelimb and hindlimb fibers of the corticospinal tract. Recognition of this syndrome might prevent inappropriate operative intervention in patients with coexisting pathological conditions of the lower cervical spinal cord.

The crystal structure of the lysyl-tRNA synthetase (LysU) from Escherichia coli.

BACKGROUND: Lysyl-tRNA synthetase catalyzes the attachment of the amino acid lysine to the cognate tRNA. The enzyme is a member of the class II amino-acyl-tRNA synthetases; the crystal structures of the seryl- and aspartyl-tRNA synthetases from this class are already known. Lysyl-tRNA synthetase shows extensive sequence homology with aspartyl-tRNA synthetase. In Escherichia coli there are two isoforms of the enzyme, LysS and LysU. Unlike LysS, which is synthesized under normal growth conditions, LysU is the product of a normally silent gene which is overexpressed under extreme physiological conditions (such as heat-shock), and can synthesize a number of adenyl dinucleotides (in particular AppppA). These dinucleotides have been proposed to act as modulators of the heat-shock response and stress response. RESULTS: The crystal structure of E. coli LysU has been determined to 2.8 A resolution, with lysine bound to the active site. The protein is a homodimer, with a rather extended dimer interface spanning the entire length of the molecule. Each monomer consists of two domains: a smaller N-terminal domain which binds the tRNA anticodon, and a larger C-terminal domain with the topology characteristic of the catalytic domain found in class II synthetases. CONCLUSIONS: A comparison of the LysU crystal structure with the structures of seryl- and aspartyl-tRNA synthetases enables a conserved core to be identified. The structural homology with the aspartyl-tRNA synthetase extends to include the anticodon-binding domain. When the active sites of lysyl-, aspartyl- and seryl-tRNA synthetases are compared, a number of catalytically important residues are conserved and a similar extended network of hydrogen bonds can be observed in the amino acid binding pocket in all three structures, although the details may differ. The lysine substrate is involved in an extended network of hydrogen bonds and polar interactions, with the side chain amino group forming a salt bridge with Glu428. The binding of ATP to LysU can be modelled on the basis of the aspartyl-tRNA synthetase-ATP complex, but the tRNA acceptor stem interaction for LysU cannot be easily modelled by similar extrapolation.

Crystallization and preliminary diffraction studies of Escherichia coli lysyl-tRNA synthetase (LysU).

Crystals of Escherichia coli lysyl-tRNA synthetase (lysU gene product) have been obtained by vapour diffusion techniques. Three different crystal forms could be grown under similar conditions. The crystals that have been chosen for the structure determination belong to space group C222(1) with cell dimensions a = 144.3 A, b = 257.8 A, c = 182.1 A and contain three monomers in the asymmetric unit. They diffract to at least 2.1 A resolution, but are very sensitive to radiation damage.