Structural characterization of the Saccharomyces cerevisiae THO complex by small-angle X-ray scattering.

The THO complex participates during eukaryotic mRNA biogenesis in coupling transcription to formation and nuclear export of translation-competent messenger ribonucleoprotein particles. In Saccharomyces cerevisiae, THO has been defined as a heteropentamer composed of the Tho2p, Hpr1p, Tex1p, Mft1p, and Thp2p subunits and the overall three-dimensional shape of the complex has been established by negative stain electron microscopy. Here, we use small-angle X-ray scattering measured for isolated THO components (Mft1p and Thp2p) as well as THO subcomplexes (Mft1p-Thp2p and Mft1p-Thp2p-Tho2p) to construct structural building blocks that allow positioning of each subunit within the complex. To accomplish this, the individual envelopes determined for Mft1p and Thp2p are first fitted inside those of the Mft1p-Thp2p and Mft1p-Thp2p-Tho2p complexes. Next, the ternary complex structure is placed in the context of the five-component electron microscopy structure. Our model reveals not only the position of each protein in the THO complex relative to each other, but also shows that the pentamer is likely somewhat larger than what was observed by electron microscopy.

Mutational analysis of the yeast RNA helicase Sub2p reveals conserved domains required for growth, mRNA export, and genomic stability.

Sub2p/UAP56 is a highly conserved DEAD-box RNA helicase involved in the packaging and nuclear export of mRNA/protein particles (mRNPs). In Saccharomyces cerevisiae, Sub2p is recruited to active chromatin by the pentameric THO complex and incorporated into the larger transcription-export (TREX) complex. Sub2p also plays a role in the maintenance of genome integrity as its inactivation causes severe transcription-dependent recombination of DNA. Despite the central role of Sub2p in early mRNP biology, little is known about its function. Here, we report the presence of an N-terminal motif (NTM) conserved specifically in the Sub2p branch of RNA helicases. Mutation of the NTM causes nuclear accumulation of poly(A)(+) RNA and impaired growth without affecting core helicase functions. Thus, the NTM functions as an autonomous unit. Moreover, two sub2 mutants, that are deficient in ATP binding, act in a trans-dominant negative fashion for growth and induce high recombination rates in vivo. Although wild-type Sub2p is prevented access to transcribed loci in such a background, this does not mechanistically explain the phenotype.

Functional consequences of T-stem mutations in E. coli tRNAThrUGU in vitro and in vivo.

The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNA(Thr)(UGU) were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound to mutations in three T-stem base pairs in a quantitatively identical manner. However, tRNA(Thr) differs from other tRNAs by also using its rare A52-C62 pair as a negative specificity determinant. Using a plasmid-based tRNA gene replacement strategy, we show that many of the tRNA(Thr)(UGU) T-stem changes are either unable to support growth of E. coli or are less effective than the wild-type sequence. Since the inviable T-stem sequences are often present in other E. coli tRNAs, it appears that T-stem sequences in each tRNA body have evolved to optimize function in a different way. Although mutations of tRNA(Thr) can substantially increase or decrease its affinity to EF-Tu, the observed affinities do not correlate with the growth phenotype of the mutations in any simple way. This may either reflect the different conditions used in the two assays or indicate that the T-stem mutants affect another step in the translation mechanism.

Exploring the specificity of bacterial elongation factor Tu for different tRNAs.

In order to identify amino acids in Thermus thermophilus elongation factor Tu which contribute to its specificity for different tRNAs, the binding affinities of 20 point mutations were compared to that of wild type protein using four tRNAs of differing affinities. The observed specificity for tRNA is the result of the varying contributions of five amino acids which make contacts with the T-stem and the 3' terminus of tRNA. For three of the amino acids the test tRNAs differ in sequence at the site of contact, presumably explaining the specificity. However, the remaining two amino acids contact tRNA at conserved positions, suggesting that more global structural or dynamic properties of the free tRNA contribute to specificity.

The 51-63 base pair of tRNA confers specificity for binding by EF-Tu.

Elongation factor Tu (EF-Tu) exhibits significant specificity for the different elongator tRNA bodies in order to offset its variable affinity to the esterified amino acid. Three X-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of tRNA, a single hydrogen bond is observed between the Glu390 and the amino group of a guanine in the 51-63 base pair in the T-stem of tRNA. Here we show that the Glu390Ala mutation of Thermus thermophilus EF-Tu selectively destabilizes binding of those tRNAs containing a guanine at either position 51 or 63 and that mutagenesis of the 51-63 base pair in several tRNAs modulates their binding affinities to EF-Tu. A comparison of Escherichia coli tRNA sequences suggests that this specificity mechanism is conserved across the bacterial domain. While this contact is an important specificity determinant, it is clear that others remain to be identified.

Directed mutagenesis identifies amino acid residues involved in elongation factor Tu binding to yeast Phe-tRNAPhe.

The co-crystal structure of Thermus aquaticus elongation factor Tu.guanosine 5'- [beta,gamma-imido]triphosphate (EF-Tu.GDPNP) bound to yeast Phe-tRNA(Phe) reveals that EF-Tu interacts with the tRNA body primarily through contacts with the phosphodiester backbone. Twenty amino acids in the tRNA binding cleft of Thermus Thermophilus EF-Tu were each mutated to structurally conservative alternatives and the affinities of the mutant proteins to yeast Phe-tRNA(Phe) determined. Eleven of the 20 mutations reduced the binding affinity from fourfold to >100-fold, while the remaining ten had no effect. The thermodynamically important residues were spread over the entire tRNA binding interface, but were concentrated in the region which contacts the tRNA T-stem. Most of the data could be reconciled by considering the crystal structures of both free EF-Tu.GTP and the ternary complex and allowing for small (1.0 A) movements in the amino acid side-chains. Thus, despite the non-physiological crystallization conditions and crystal lattice interactions, the crystal structures reflect the biochemically relevant interaction in solution.

The affinity of elongation factor Tu for an aminoacyl-tRNA is modulated by the esterified amino acid.

When different mutations were introduced into the anticodon loop and at position 73 of YFA2, a derivative of yeast tRNA(Phe), a single tRNA body was misacylated with 13 different amino acids. The affinities of these misacylated tRNAs for Thermus thermophilus elongation factor Tu (EF-Tu).GTP were determined using a ribonuclease protection assay. A range of 2.5 kcal/mol in the binding energies was observed, clearly demonstrating that EF-Tu specifically recognizes the side chain of the esterified amino acid. Furthermore, this specificity can be altered by introducing a mutation in the amino acid binding pocket on the surface of EF-Tu. Also, when discussed in conjunction with the previously determined specificity of EF-Tu for the tRNA body, these experiments further demonstrate that EF-Tu uses thermodynamic compensation to bind cognate aminoacyl-tRNAs similarly.

The 1987= mil novecientos ochenta y siete forest fire disaster in California : Assessment of emergency room visits

During a 5-d period that commenced on August 30, 1987, dry lightning strikes ignited more than 1500 fires that destroyed in excess of 600000 acres of California forests. To evaluate the public health impact of the smoke on the general population, all hospital emergency rooms located in the six counties most sevely sffected by smoke or fire were surveyed. Selected hospital information was abstracted for a 2 1/2-wk period during the fires and during two reference periodds. During the period of major forest fire activity, visits of persons with asthma and chronic obstructive pulmonary disease increased in number (observed/expected ratios of 1.4 and 1.3, respectively), as did visits of persons with sinusitis, upper respiratoey infections, and laryngitis. A few patients with acute respiratory or eye irritation also visited the emergency rooms. Even recognizing the limited sensitivity of emergency room surveys, the overall public health impact was relatively modest. The increased respiratory moribidity detected in this survey, however, supports the notion that persons with pre-existing respiratory disease represent a sensitive subpopulation, who should be targeted for purposes of public health intervention when exposure to forest fire smoke is likely.(AU)

The public health consequences of disasters : 1989

The contents of the report are divided into several major sections: general concerns, geophysical events, weather-related problems, and human-generated problems. The first chapter, a kind of primer, describes the concepts and role of surveillance and epidemiology-the database for and the science of public health practice, respectively. The second chapter very briefly discusses some important considerations relating to communications efforts to communications efforts between health officials and the news media in times of disaster. The last chapter in this section addresses communicable disease control following natural disasters

The other chapters, which cover discrete types of disasters, emphasize such areas as the history and nature of the disasters, as well as causative factors for the natural disasters that may influence morbidity and mortality. Next, the report describes the public health implications of such events including a) prevention and control measures, b) surveillance, and c) research recommendations in areas in which the public health practitioner needs more useful information

Surveillance for adverse health effects following a chemical release in West Virginia

On 11 August 1985, a large inadvertent release occurred from a chemical manufacturing plant in Institute, West Virginia. The emission was related to overheating of a storage tank that contained chemicals for the production of aldicarb, a cholinesterase inhibiting pesticide. As a result of this emission, 136 persons were treated in five local emergency romms, 29 of whom were hospitalized for one or more days, mostly for observation. No fatalities resulted. Interviews with treated persons and non-treated community residents confirmed that the health effects were transient and compatible with exposure to irritating vapors rather than with exposure to methyl isocyanate (a chemical used in the process) or to aldicarb. Only 5 per cent of the treated persons and 5 per cent of the community residents surveyed were adequately warned of the emission by the plant siren. Because persons in the nearby community are potentially vulnerable to chemical injury, we recommend the development of an improved warning system, a formal evacuation procedure, and other measures to limit exposure of persons in this area in the event of a future release