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T Pape

Publications and source records attributed to T Pape.

17 recordsLinked to original sources
PubMed2006

A second paradigm for gene activation in bacteria.

Control of gene expression is key to development and adaptation. Using purified transcription components from bacteria, we employ structural and functional studies in an integrative manner to elaborate a detailed description of an obligatory step, the accessing of the DNA template, in gene expression. Our work focuses on a specialized molecular machinery that utilizes ATP hydrolysis to initiate DNA opening and permits a description of how the events triggered by ATP hydrolysis within a transcriptional activator can lead to DNA opening and transcription. The bacterial EBPs (enhancer binding proteins) that belong to the AAA(+) (ATPases associated with various cellular activities) protein family remodel the RNAP (RNA polymerase) holoenzyme containing the sigma(54) factor and convert the initial, transcriptionally silent promoter complex into a transcriptionally proficient open complex using transactions that reflect the use of ATP hydrolysis to establish different functional states of the EBP. A molecular switch within the model EBP we study [called PspF (phage shock protein F)] is evident, and functions to control the exposure of a solvent-accessible flexible loop that engages directly with the initial RNAP promoter complex. The sigma(54) factor then controls the conformational changes in the RNAP required to form the open promoter complex.

Bacteria↗
PubMed2005

Hypoderma sinense: solving a century-old enigma.

Among the species of Hypoderma (Diptera: Oestridae) that have been described and named over the last three centuries, Hypoderma sinense Pleske has been the subject of several scientific discussions. Hypoderma sinense was described by T. Pleske in 1926 on the basis of only three females collected by the Russian explorer P. K. Kozlov nearly 25 years earlier during a scientific expedition to China (1900-1901). This species was examined by the foremost oestrid authorities and synonomized with H. lineatum. Recently a unique, unidentified species of Hypoderma was observed to infect cattle and yaks in China. Molecular and morphological observations confirmed the unique nature of the third-stage larvae. This data initiated a debate within the scientific community regarding the proper name of this species, in particular with reference to previous taxonomical discussion on the validity of H. sinense. The present work provides a historical overview of the Russian scientific expeditions that collected the specimens and of the explorers and the entomologists who contributed to the description of H. sinense. The morphological examination of the original type material of H. sinense and the comparison with females of H. lineatum indicated that the H. sinense lectotype, deposited at the Zoological Institute of the Russian Academy of Sciences, St Petersburg, was within the range of variation of H. lineatum. Comparisons of the cox1 (688 bp) sequence obtained from the leg of a paralectotype of H. sinense with those of H. bovis (Linneaus), H. lineatum (De Villers) and of a sixth valid species of Hypoderma identified as "H. sinense" available in GenBank revealed differences of 9.7%, 7.2% and 0.3%, respectively. On the basis of these results, we concluded that the nominal species H. sinense should be treated as valid.

Animals↗
PubMed2001

Direct localization by cryo-electron microscopy of secondary structural elements in Escherichia coli 23 S rRNA which differ from the corresponding regions in Haloarcula marismortui.

Insertions were introduced by a two-step mutagenesis procedure into each of five double-helical regions of Escherichia coli 23 S rRNA, so as to extend the helix concerned by 17 bp. The helices chosen were at sites within the 23 S molecule (h9, h25, h45, h63 and h98) where significant length variations between different species are known to occur. At each of these positions, with the exception of h45, there are also significant differences between the 23 S rRNAs of E. coli and Haloarcula marismortui. Plasmids carrying the insertions were introduced into an E. coli strain lacking all seven rrn operons. In four of the five cases the cells were viable and 50 S subunits could be isolated; only the insertion in h63 was lethal. The modified subunits were examined by cryo-electron microscopy (cryo-EM), with a view to locating extra electron density corresponding to the insertion elements. The results were compared both with the recently determined atomic structure of H. marismortui 23 S rRNA in the 50 S subunit, and with previous 23 S rRNA modelling studies based on cryo-EM reconstructions of E. coli ribosomes. The insertion element in h45 was located by cryo-EM at a position corresponding precisely to that of the equivalent helix in H. marismortui. The insertion in h98 (which is entirely absent in H. marismortui) was similarly located at a position corresponding precisely to that predicted from the E. coli modelling studies. In the region of h9, the difference between the E. coli and H. marismortui secondary structures is ambiguous, and the extra electron density corresponding to the insertion was seen at a location intermediate between the position of the nearest helix in the atomic structure and that in the modelled structure. In the case of h25 (which is about 50 nucleotides longer in H. marismortui), no clear extra cryo-EM density corresponding to the insertion could be observed.

Base Sequence↗
PubMed2001

DNA-based identification and molecular systematics of forensically important Sarcophagidae (Diptera).

Sarcophagid flies have many characteristics that make them ideal forensic indicators. However, their utility is severely limited because it is difficult or impossible to determine the species of a sarcophagid larva, and in many instances an adult specimen, based on anatomy. We developed a database of mitochondrial DNA sequence data that makes it possible to identify all sarcophagid species likely to be found feeding on a human corpse at an urban location in Canada or the USA. Analyses were based on a 783 base pair region of the gene for cytochrome oxidase subunit one (COI). The species analyzed, including some of no forensic importance that were included for purposes of phylogenetic comparisons, were members of the genera Sarcophaga, Peckia, Blaesoxipha, Rovinia, Wohlfahrtia, Brachicoma (all Sarcophagidae), and Musca (Muscidae).

Animals↗
PubMed2000

Intact aminoacyl-tRNA is required to trigger GTP hydrolysis by elongation factor Tu on the ribosome.

GTP hydrolysis by elongation factor Tu (EF-Tu) on the ribosome is induced by codon recognition. The mechanism by which a signal is transmitted from the site of codon-anticodon interaction in the decoding center of the 30S ribosomal subunit to the site of EF-Tu binding on the 50S subunit is not known. Here we examine the role of the tRNA in this process. We have used two RNA fragments, one which contains the anticodon and D hairpin domains (ACD oligomer) derived from tRNA(Phe) and the second which comprises the acceptor stem and T hairpin domains derived from tRNA(Ala) (AST oligomer) that aminoacylates with alanine and forms a ternary complex with EF-Tu. GTP. While the ACD oligomer and the ternary complex containing the Ala-AST oligomer interact with the 30S and 50S A site, respectively, no rapid GTP hydrolysis was observed when both were bound simultaneously. The presence of paromomycin, an aminoglycoside antibiotic that binds to the decoding site and stabilizes codon-anticodon interaction in unfavorable coding situations, did not increase the rate of GTP hydrolysis. These results suggest that codon recognition as such is not sufficient for GTPase activation and that an intact tRNA molecule is required for transmitting the signal created by codon recognition to EF-Tu.

Anticodon↗
PubMed2000

Boyer's model of scholarly nursing applied to professional development.

Scholarship in nursing often is difficult to define and identify, but it is an important element in nursing. Scholars are needed in every practice setting to question the status quo and pursue fresh approaches to issues. This article examines Boyer's revised model of scholarship, which uses paradigms of discovery, integration, application, and teaching. Knowledge dissemination is emphasized as an important component for sharing information with fellow nurses and the public. Boyer's model of nursing scholarship provides institutions and OR nurse managers with a scholarly method for evaluating employee efforts. Strategies offered include professional development plans and employee portfolios. These applications encourage scholarly activities.

Diffusion of Innovation↗
PubMed2000

Conformational switch in the decoding region of 16S rRNA during aminoacyl-tRNA selection on the ribosome.

Binding of aminoglycoside antibiotics to 16S ribosomal RNA induces a particular structure of the decoding center and increases the misincorporation of near-cognate amino acids. By kinetic analysis we show that this is due to stabilization of the near-cognate codon recognition complex and the acceleration of two rearrangements that limit the rate of amino acid incorporation. The same rearrangement steps are accelerated in the cognate coding situation. We suggest that cognate codon recognition, or near-cognate codon recognition augmented by aminoglycoside binding, promote the transition of 16S rRNA from a 'binding' to a 'productive' conformation that determines the fidelity of decoding.

Anti-Bacterial Agents↗
PubMed1999

The Escherichia coli large ribosomal subunit at 7.5 A resolution.

BACKGROUND: In recent years, the three-dimensional structure of the ribosome has been visualised in different functional states by single-particle cryo-electron microscopy (cryo-EM) at 13-25 A resolution. Even more recently, X-ray crystallography has achieved resolution levels better than 10 A for the ribosomal structures of thermophilic and halophilic organisms. We present here the 7.5 A solution structure of the 50S large subunit of the Escherichia coli ribosome, as determined by cryo-EM and angular reconstitution. RESULTS: The reconstruction reveals a host of new details including the long alpha helix connecting the N- and C-terminal domains of the L9 protein, which is found wrapped like a collar around the base of the L1 stalk. A second L7/L12 dimer is now visible below the classical L7/L12 'stalk', thus revealing the position of the entire L8 complex. Extensive conformational changes occur in the 50S subunit upon 30S binding; for example, the L9 protein moves by some 50 A. Various rRNA stem-loops are found to be involved in subunit binding: helix h38, located in the A-site finger; h69, on the rim of the peptidyl transferase centre cleft; and h34, in the principal interface protrusion. CONCLUSIONS: Single-particle cryo-EM is rapidly evolving towards the resolution levels required for the direct atomic interpretation of the structure of the ribosome. Structural details such as the minor and major grooves in rRNA double helices and alpha helices of the ribosomal proteins can already be visualised directly in cryo-EM reconstructions of ribosomes frozen in different functional states.

Bacterial Proteins↗
PubMed1999

Induced fit in initial selection and proofreading of aminoacyl-tRNA on the ribosome.

The fidelity of aminoacyl-tRNA (aa-tRNA) selection by the bacterial ribosome is determined by initial selection before and proofreading after GTP hydrolysis by elongation factor Tu. Here we report the rate constants of A-site binding of a near-cognate aa-tRNA. The comparison with the data for cognate aa-tRNA reveals an additional, important contribution to aa-tRNA discrimination of conformational coupling by induced fit. It is found that two rearrangement steps that limit the chemical reactions of A-site binding, i.e. GTPase activation (preceding GTP hydrolysis) and A-site accommodation (preceding peptide bond formation), are substantially faster for cognate than for near-cognate aa-tRNA. This suggests an induced-fit mechanism of aa-tRNA discrimination on the ribosome that operates in both initial selection and proofreading. It is proposed that the cognate codon-anticodon interaction, more efficiently than the near-cognate one, induces a particular conformation of the decoding center of 16S rRNA, which in turn promotes GTPase activation and A-site accommodation of aa-tRNA, thereby accelerating the chemical steps. As kinetically favored incorporation of the correct substrate has also been suggested for DNA and RNA polymerases, the present findings indicate that induced fit may contribute to the fidelity of template-programed systems in general.

Anticodon↗
PubMed1999

A systems approach to resolving OR conflict.

Effective communication and courteous interaction with other hospital departments are essential for a smooth-running OR. To function effectively in a perioperative setting, OR personnel need patience, knowledge, and skill in problem-solving techniques. When health care employees are not taught these techniques and protocols are not in place to manage conflict, friction develops and productivity suffers. In this article, conflict situations between two hospital departments and on OR are presented to characterize successful conflict resolution methods using a systems approach. The results include reduction of surgical procedure delays, efficient ordering of supplies, and improved interpersonal relations.

Conflict, Psychological↗
PubMed1998

Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.

The kinetic mechanism of elongation factor Tu (EF-Tu)-dependent binding of Phe-tRNAPhe to the A site of poly(U)-programmed Escherichia coli ribosomes has been established by pre-steady-state kinetic experiments. Six steps were distinguished kinetically, and their elemental rate constants were determined either by global fitting, or directly by dissociation experiments. Initial binding to the ribosome of the ternary complex EF-Tu.GTP.Phe-tRNAPhe is rapid (k1 = 110 and 60/micromM/s at 10 and 5 mM Mg2+, 20 degreesC) and readily reversible (k-1 = 25 and 30/s). Subsequent codon recognition (k2 = 100 and 80/s) stabilizes the complex in an Mg2+-dependent manner (k-2 = 0.2 and 2/s). It induces the GTPase conformation of EF-Tu (k3 = 500 and 55/s), instantaneously followed by GTP hydrolysis. Subsequent steps are independent of Mg2+. The EF-Tu conformation switches from the GTP- to the GDP-bound form (k4 = 60/s), and Phe-tRNAPhe is released from EF-Tu.GDP. The accommodation of Phe-tRNAPhe in the A site (k5 = 8/s) takes place independently of EF-Tu and is followed instantaneously by peptide bond formation. The slowest step is dissociation of EF-Tu.GDP from the ribosome (k6 = 4/s). A characteristic feature of the mechanism is the existence of two conformational rearrangements which limit the rates of the subsequent chemical steps of A-site binding.

Codon↗
PubMed1997

Legal and ethical considerations of informed consent.

The law of informed consent remains ineffective at resolving patient comprehension issues primarily because differing interpretations exist regarding who is responsible for the duty to inform. Court cases continue to set precedents for practicing physicians and other health care providers; however, other measures can be applied for effectual patient advocacy. Health care personnel should rewrite typical consent forms in simpler terms, use larger print, and create duplicate copies. If patients are given copies of the permits they sign, the can reread the forms at home when they are more comfortable. For true autonomy to exist in informed consent for surgical procedures, consent forms should contain patients' primary languages whenever possible, or an adequate interpreter should be made available. Surgeons, nurses, and other health care providers must become aware of their responsibilities related to informed consent for treatment. It is necessary for health care personnel to develop and use effective communication techniques and remember that although some patients are more visually attuned to new information, other patients may benefit more from listening or reading. The cases in this article show that a patient's autonomy is part of the informed consent process and the duty to inform the patient lies with the person performing the procedure. A more important issue, however, involves the patient's comprehension of the information given, because without it, the patient cannot achieve true autonomy in making decisions. Ensuring that all elements of informed consent are met to obtain informed consent will result in fewer malpractice claims, greater patient satisfaction, and an improved professional image. Nevertheless, nurses should make themselves aware of the state laws in which they practice, including their nurse practice acts. They then should advocate for patient rights by encompassing all elements of informed consent.

Ethics, Medical↗
PubMed1996

The G222D mutation in elongation factor Tu inhibits the codon-induced conformational changes leading to GTPase activation on the ribosome.

Elongation factor Tu (EF-Tu) from Escherichia coli carrying the mutation G222D is unable to hydrolyze GTP on the ribosome and to sustain polypeptide synthesis at near physiological Mg2+ concentration, although the interactions with guanine nucleotides and aminoacyl-tRNA are not changed significantly. GTPase and polypeptide synthesis activities are restored by increasing the Mg2+ concentration. Here we report a pre-steady-state kinetic study of the binding of the ternary complexes of wild-type and mutant EF-Tu with Phe-tRNA(Phe) and GTP to the A site of poly(U)-programed ribosomes. The kinetic parameters of initial binding to the ribosome and subsequent codon-anticodon interaction are similar for mutant and wild-type EF-Tu, independent of the Mg2+ concentration, suggesting that the initial interaction with the ribosome is not affected by the mutation. Codon recognition following initial binding is also not affected by the mutation. The main effect of the G222D mutation is the inhibition, at low Mg2+ concentration, of codon-induced structural transitions of the tRNA and, in particular, their transmission to EF-Tu that precedes GTP hydrolysis and the subsequent steps of A-site binding. Increasing the Mg2+ concentration to 10 mM restores the complete reaction sequence of A-site binding at close to wild-type rates. The inhibition of the structural transitions is probably due to the interference of the negative charge introduced by the mutation with negative charges either of the 3' terminus of the tRNA, bound in the vicinity of the mutated amino acid in domain 2 of EF-Tu, or of the ribosome. Increasing the Mg2+ concentration appears to overcome the inhibition by screening the negative charges.

Codon↗
PubMed1996

Initial binding of the elongation factor Tu.GTP.aminoacyl-tRNA complex preceding codon recognition on the ribosome.

The first step in the sequence of interactions between the ribosome and the complex of elongation factor Tu (EF-Tu), GTP, and aminoacyl-tRNA, which eventually leads to A site-bound aminoacyl-tRNA, is the codon-independent formation of an initial complex. We have characterized the initial binding and the resulting complex by time-resolved (stopped-flow) and steady-state fluorescence measurements using several fluorescent tRNA derivatives. The complex is labile, with rate constants of 6 x 10(7) M-1 s-1 and 24 s-1 (20 degrees C, 10 mM Mg2+) for binding and dissociation, respectively. Both thermodynamic and activation parameters of initial binding were determined, and five Mg2+ ions were estimated to participate in the interaction. While a cognate ternary complex proceeds form initial binding through codon recognition to rapid GTP hydrolysis, the rate constant of GTP hydrolysis in the non-cognate complex is 4 orders of magnitude lower, despite the rapid formation of the initial complex in both cases. Hence, the ribosome-induced GTP hydrolysis by EF-Tu is strongly affected by the presence of the tRNA. This suggests that codon-anticodon recognition, which takes place after the formation of the initial binding complex, provides a specific signal that triggers fast GTP hydrolysis by EF-Tu on the ribosome.

Codon↗
PubMedDate not supplied

Elongation factor Tu, a GTPase triggered by codon recognition on the ribosome: mechanism and GTP consumption.

The mechanism of elongation factor Tu (EF-Tu) catalyzed aminoacyl-tRNA (aa-tRNA) binding to the A site of the ribosome was studied. Two types of complexes of EF-Tu with GTP and aa-tRNA, EF-Tu.GTP-aa-tRNA (ternary) and (EF-Tu.GTP)2.aa-tRNA (quinternary), can be formed in vitro depending on the conditions. On interaction with the ribosomal A site, generally only one molecule of GTP is hydrolysed per aa-tRNA bound and peptide bond formed. The second GTP molecule from the quinternary complex is hydrolyzed only during translation of an oligo(U) tract in the presence of EF-G. The first step in the interaction between the ribosome and the ternary complex is the codon-independent formation of an initial complex. In the absence of codon recognition, the aa-tRNA-EF-Tu complex does not enter further steps of A site binding and remains in the initial binding state. Despite the rapid formation of the initial complex, the rate constant of GTP hydrolysis in the noncognate complex is four orders of magnitude lower compared with the cognate complex. This, together with the results of time-resolved fluorescence measurements, suggests that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements that result in a conformational change of EF-Tu, presumably involving the effector region, which, in turn, triggers GTP hydrolysis and the subsequent steps of A site binding.

Base Sequence↗