Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase.

Analysis of prolyl-tRNA synthetase (ProRS) across all three taxonomic domains (Eubacteria, Eucarya, and Archaea) reveals that the sequences are divided into two distinct groups. Recent studies show that Escherichia coli ProRS, a member of the "prokaryotic-like" group, recognizes specific tRNA bases at both the acceptor and anticodon ends, whereas human ProRS, a member of the "eukaryotic-like" group, recognizes nucleotide bases primarily in the anticodon. The archaeal Methanococcus jannaschii ProRS is a member of the eukaryotic-like group, although its tRNA(Pro) possesses prokaryotic features in the acceptor stem. We show here that, in some respects, recognition of tRNA(Pro) by M. jannaschii ProRS parallels that of human, with a strong emphasis on the anticodon and only weak recognition of the acceptor stem. However, our data also indicate differences in the details of the anticodon recognition between these two eukaryotic-like synthetases. Although the human enzyme places a stronger emphasis on G35, the M. jannaschii enzyme places a stronger emphasis on G36, a feature that is shared by E. coli ProRS. These results, interpreted in the context of an extensive sequence alignment, provide evidence of divergent adaptation by M. jannaschii ProRS; recognition of the tRNA acceptor end is eukaryotic-like, whereas the details of the anticodon recognition are prokaryotic-like. This divergence may be a reflection of the unusual dual function of this enzyme, which catalyzes specific aminoacylation with proline as well as with cysteine.

Alternative design of a tRNA core for aminoacylation.

The core of Escherichia coli tRNA(Cys) is important for aminoacylation of the tRNA by cysteine-tRNA synthetase. This core differs from the common tRNA core by having a G15:G48, rather than a G15:C48 base-pair. Substitution of G15:G48 with G15:C48 decreases the catalytic efficiency of aminoacylation by two orders of magnitude. This indicates that the design of the core is not compatible with G15:C48. However, the core of E. coli tRNA(Gln), which contains G15:C48, is functional for cysteine-tRNA synthetase. Here, guided by the core of E. coli tRNA(Gln), we sought to test and identify alternative functional design of the tRNA(Cys) core that contains G15:C48. Although analysis of the crystal structure of tRNA(Cys) and tRNA(Gln) implicated long-range tertiary base-pairs above and below G15:G48 as important for a functional core, we showed that this was not the case. The replacement of tertiary interactions involving 9, 21, and 59 in tRNA(Cys) with those in tRNA(Gln) did not construct a functional core that contained G15:C48. In contrast, substitution of nucleotides in the variable loop adjacent to 48 of the 15:48 base-pair created functional cores. Modeling studies of a functional core suggests that the re-constructed core arose from enhanced stacking interactions that compensated for the disruption caused by the G15:C48 base-pair. The repacked tRNA core displayed features that were distinct from those of the wild-type and provided evidence that stacking interactions are alternative means than long-range tertiary base-pairs to a functional core for aminoacylation.

Influence of transfer RNA tertiary structure on aminoacylation efficiency by glutaminyl and cysteinyl-tRNA synthetases.

The position of the tertiary Levitt pair between nucleotides 15 and 48 in the transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops. E. coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairing at this position (G15-C48), while the tRNA(Cys) species from this organism instead features an unusual G15-G48 pair. To explore the structural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates for glutaminyl and cysteinyl-tRNA synthetases. The glutaminylation efficiencies of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of this tRNA plays a role in GlnRS recognition. Introduction of tRNA(Cys) identity nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair. Surprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS recognition. By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G15-G48 with much better efficiency than those with G15-C48. Therefore, tertiary nucleotides surrounding the Levitt pair must significantly modulate the efficiency of aminoacylation by CysRS. To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS. These structures show that the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from several groups of G15. The G15-G48 conformations differ significantly compared to that observed in the native tRNA(Cys) structure bound to EF-Tu, further implicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present crucial recognition determinants to aminoacyl-tRNA synthetases.

Synthesis of cysteinyl-tRNA(Cys) by a genome that lacks the normal cysteine-tRNA synthetase.

Synthesis of cysteinyl-tRNA(Cys) by cysteine-tRNA synthetase is required for decoding cysteine codons in all known organisms. The genome of the archaeon Methanococcus jannaschii lacks the gene for a normal cysteine-tRNA synthetase. The activity of the enzyme, however, was identified recently, and it allowed the purification of the enzyme and cloning of its gene. Sequence analysis of the gene showed that it encodes proline-tRNA synthetase and, thus, raised the possibility of dual activities in a single aminoacyl-tRNA synthetase. Assays of aminoacyl-adenylate synthesis confirmed the ability of the enzyme to activate proline and cysteine and showed that both activities were independent of tRNA. Assays of tRNA aminoacylation established the specific attachment of proline to tRNA(Pro) and cysteine to tRNA(Cys). However, in contrast to a recent report of comparable activities with cysteine and proline, results here indicate that the adenylate synthesis and aminoacylation activities with cysteine are significantly lower than the respective activity with proline. In addition, there is evidence of overlapping amino acid-binding sites and tRNA-binding sites. These considerations, among others, raised the distinct possibility that the M. jannaschii proline-tRNA synthetase may recruit additional protein or RNA factors to facilitate the synthesis of cysteinyl-tRNA(Cys).

Evidence for unfolding of the single-stranded GCCA 3'-End of a tRNA on its aminoacyl-tRNA synthetase from a stacked helical to a foldback conformation.

The conformation of a tRNA in its initial contact with its cognate aminoacyl-tRNA synthetase was investigated with the Escherichia coli glutamyl-tRNA synthetase-tRNA(Glu) complex. Covalent complexes between the periodate-oxidized tRNA(Glu) and its synthetase were obtained. These complexes are specific since none were formed with any other oxidized E. coli tRNA. The three major residues cross-linked to the 3'-terminal adenosine of oxidized tRNA(Glu) are Lys115, Arg209, and Arg48. Modeling of the tRNA(Glu)-glutamyl-tRNA synthetase based on the known crystal structures of Thermus thermophilus GluRS and of the E. coli tRNA(Gln)-glutaminyl-tRNA synthetase complex shows that these three residues are located in the pocket that binds the acceptor stem, and that Lys115, located in a 26 residue loop closed by coordination to a zinc atom in the tRNA acceptor stem-binding domain, is the first contact point of the 3'-terminal adenosine of tRNA(Glu). In our model, we assume that the 3'-terminal GCCA single-stranded segment of tRNA(Glu) is helical and extends the stacking of the acceptor stem. This assumption is supported by the fact that the 3' CCA sequence of tRNA(Glu) is not readily circularized in the presence of T4 RNA ligase under conditions where several other tRNAs are circularized. The two other cross-linked sites are interpreted as the contact sites of the 3'-terminal ribose on the enzyme during the unfolding and movement of the 3'-terminal GCCA segment to position the acceptor ribose in the catalytic site for aminoacylation.

Conservation of a tRNA core for aminoacylation.

The core region of Escherichia coli tRNA(Cys)is important for aminoacylation of the tRNA. This core contains an unusual G15:G48 base pair, and three adenosine nucleotides A13, A22 and A46 that are likely to form a 46:[13:22] adenosine base triple. We recently observed that the 15:48 base pair and the proposed 46:[13:22] triple are structurally and functionally coupled to contribute to aminoacylation. Inspection of a database of tRNA sequences shows that these elements are only found in one other tRNA, the Haemophilus influenzae tRNA(Cys). Because of the complexity of the core, conservation of sequence does not mean conservation of function. We here tested whether the conserved elements in H. influenzae tRNA(Cys)were also important for aminoacylation of H. influenzae tRNA(Cys). We cloned and purified a recombinant H. influenzae cysteine-tRNA synthe-tase and showed that it depends on 15:48 and 13, 22 and 46 in a relationship analogous to that of E. coli cysteine-tRNA synthetase. The functional conservation of the tRNA core is correlated with sequence conservation between E.coli and H.influenzae cysteine-tRNA synthetases. As the genome of H. influenzae is one of the smallest and may approximate a small autonomous entity in the development of life, the dependence of this genome on G15:G48 and its coupling with the proposed A46:[A13:A22] triple for aminoacylation with cysteine suggests an early role of these motifs in the evolution of decoding genetic information.

An archaeal aminoacyl-tRNA synthetase missing from genomic analysis.

The complete genomic sequencing of Methanococcus jannaschii cannot identify the gene for the cysteine-specific member of aminoacyl-tRNA synthetases. However, we show here that enzyme activity is present in the cell lysate of M. jannaschii. The demonstration of this activity suggests a direct pathway for the synthesis of cysteinyl-tRNA(Cys) during protein synthesis.

Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases catalyze aminoacylation of tRNAs by joining an amino acid to its cognate tRNA. The selection of the cognate tRNA is jointly determined by separate structural domains that examine different regions of the tRNA. The cysteine-tRNA synthetase of Escherichia coli has domains that select for tRNAs containing U73, the GCA anticodon, and a specific tertiary structure at the corner of the tRNA L shape. The E. coli enzyme does not efficiently recognize the yeast or human tRNACys, indicating the evolution of determinants for tRNA aminoacylation from E. coli to yeast to human and the coevolution of synthetase domains that interact with these determinants. By successively modifying the yeast and human tRNACys to ones that are efficiently aminoacylated by the E. coli enzyme, we have identified determinants of the tRNA that are important for aminoacylation but that have diverged in the course of evolution. These determinants provide clues to the divergence of synthetase domains. We propose that the domain for selecting U73 is conserved in evolution. In contrast, we propose that the domain for selecting the corner of the tRNA L shape diverged early, after the separation between E. coli and yeast, while that for selecting the GCA-containing anticodon loop diverged late, after the separation between yeast and human.

An unnatural folate stereoisomer is catalytically competent in DNA photolyase.

The folate chromophore in native Escherichia coli DNA photolyase ([6R]-5,10-CH+-H4Pte(Glu)n=3-6) serves as an antenna, transferring light energy to the fully reduced flavin (FADH2) reaction center at high efficiency (EET = 0.92). Apophotolyase reconstituted after an overnight incubation with [6R,S]-5,10-CH+-H4folate (a monoglutamate analogue of the native cofactor) contains equimolar amounts of the [6R]- and [6S]-isomers, suggesting similar binding affinities. A rapid, biphasic increase in fluorescence (approximately 100-fold) is observed upon binding of 5,10-CH+-H4folate to apophotolyase at 5 degrees C; the [6S]-isomer binds about 25-fold faster than the [6R]-isomer. Although identical absorption and fluorescence emission maxima are observed for enzyme reconstituted with [6S]-, [6R]-, or [6R,S]-5,10-CH+-H4folate, folate fluorescence quantum yield values vary depending on the stereochemical configuration at the 6 position (theta = 0.18, 0.82, or 0.46, respectively, at 5 degrees C), a feature not seen with free folate. The fluorescence of enzyme-bound folate is quenched upon flavin binding; the efficiency of quenching by flavin radical (EQ = 0.96) or FADH2 (EQ = 0.89) is the same for both folate isomers. In contrast, energy transfer from folate to FADH2 is sensitive to the stereochemical configuration at the 6 position. The efficiency of energy transfer observed for enzyme containing FADH2 and [6S]-, [6R]-, or [6R,S]-5,10-CH+-H4folate (theta = 0.26, 0.66, or 0.44, respectively) is directly proportional to the fluorescence quantum yield observed for folate in the absence of FADH2, as expected for Förster-type energy transfer. Although less efficient, the unnatural [6S]-isomer is catalytically functional, a feature not previously observed with other folate-dependent enzymes. Fluorescence quantum yield studies at 77 K with free (theta = 0.67) and enzyme-bound (theta = 1.0) folate suggest that differences in solvent exposure may contribute to the fluorescence efficiency differences observed with the enzyme-bound folate isomers at 5 degrees C.

Stereospecificity of folate binding to DNA photolyase from Escherichia coli.

DNA photolyase from Escherichia coli contains folate ([6S]-5,10-CH(+)-H4Pte(Glu)n = 3-6) and reduced FAD. The folate chromophore acts as an antenna, harvesting light energy which is transferred to the reduced flavin where DNA repair occurs. The folate binding stereospecificity of the enzyme was investigated by reconstituting the apoenzyme with [6R,S]-5,10-CH(+)-H4folate and reduced FAD. The isomer composition of [methyl-3H]-5-CH3-H4folate, released into solution upon reduction of the reconstituted enzyme with [3H]NaBH4, was analyzed by enzymatic and chiral chromatographic methods. Both methods showed that the reconstituted enzyme contained nearly equimolar amounts of [6R]- and [6S]-5,10-CH(+)-H4folate.

Cognitive performance as modified by age and ECT history.

1. Cognitive processing, as measured by 15 subtest from the Wechsler Memory Scale (WMS) and the Wechsler Adult Intelligence Scale-Revised (WAIS-R) was assessed in 109 depressed inpatients who were classified as under age 65 (N = 54) or 65 or older (N = 55). Within each age group, patients were further classified according to whether they had no prior ECT (N = 40), one prior ECT series (N = 37), or two or more prior series of ECT (N = 32). 2. After examining the data set for possible sources of bias such as group differences in the severity of depression, time since last ECT series, unilateral vs. bilateral electrode placement, adjunctive use of psychotropic medication, gender, education and so forth, a MANCOVA and a series of 15 two (age) by three (ECT history) by two (gender) analyses of covariance (education served as the covariate) were used to examine the main effects of ECT history, age and their interaction on cognitive performance. 3. No generalized adverse effects of prior ECT treatment were found but older patients with two or more prior ECT series performed significantly more poorly than other subgroups on 4 of 15 cognitive tests: (1) Personal and Current Information from the WMS, (2) WMS Stories recall, (3) 30-minute delayed WMS Stories and (4) WAIS-R Similarities. 4. These findings suggest that the verbal-narrative memory functioning of depressed geriatric patients with a history of at least one prior ECT series is particularly vulnerable to disruption.

Direct evidence for singlet-singlet energy transfer in Escherichia coli DNA photolyase.

The active form of native Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate [5,10-CH(+)-H4Pte(Glu)n]. Enzyme containing FADH2 and/or 5,10-methyltetrahydrofolate (5,10-CH(+)-H4folate) can be prepared in reconstitution experiments. Fluorescence quantum yield measurements at various wavelengths with native or reconstituted enzyme provide a simple method for detecting singlet-singlet energy transfer from pterin to FADH2, a key step in the proposed catalytic mechanism. The data satisfy the following criteria: (1) Wavelength-independent quantum yield values are observed for 5,10-CH(+)-H4folate in the absence (0.434) or presence (3.57 X 10(-2)) of FADH2, for 5,10-CH(+)-H4Pte(Glu)n in the presence of FADH2 (5.58 X 10(-2)) and for FADH2 in the absence of pterin (5.34 X 10(-3)); (2) The observed decrease in pterin fluorescence quantum yield in the presence of FADH2 can be used to estimate the efficiency of pterin fluorescence quenching (EQ = 0.918 or 0.871 with 5,10-CH(+)-H4folate or 5,10-CH(+)-H4Pte(Glu)n, respectively); (3) The fluorescence quantum yield of FADH2 is increased in the presence of pterin and varies depending on the excitation wavelength, in agreement with the predicted effect of energy transfer on acceptor fluorescence quantum yield [phi acceptor (+ donor)/phi acceptor (alone) = 1 + EET(epsilon donor/epsilon acceptor), where EET is the efficiency of the energy transfer process]. With 5,10-CH(+)-H4Pte(Glu)n in native enzyme the value obtained for EET (0.92) is similar to EQ, whereas with 5,10-CH(+)-H4folate in reconstituted enzyme the value obtained for EET (0.46) is 2-fold smaller than EQ.(ABSTRACT TRUNCATED AT 250 WORDS)

Imipramine and chlordiazepoxide in depressive and anxiety disorders. I. Efficacy in depressed outpatients.

We randomly assigned 425 outpatients, independently classified as primarily depressed by two trained psychiatrists, to double-blind treatment with Imipramine hydrochloride, chlordiazepoxide hydrochloride, or placebo. Those patients who remained at least moderately depressed (following a two-week placebo washout period) were treated for an additional eight weeks. An endpoint analysis of 387 patients who completed two or more weeks of medication disclosed early therapeutic advantages of chlordiazepoxide. By week 4 of treatment, however, imipramine produced more improvement than did placebo and chlordiazepoxide. By six and eight weeks a general, marked therapeutic advantage was found for imipramine relative to placebo and to chlordiazepoxide on measures of depression, anxiety, anger-hostility, interpersonal sensitivity, and global improvement. Chlordiazepoxide-treated patients generally did significantly better on sleep difficulty but significantly worse on anger-hostility and interpersonal sensitivity than did imipramine- or placebo-treated patients.

Imipramine and chlordiazepoxide in depressive and anxiety disorders. II. Efficacy in anxious outpatients.

A double-blind, placebo-controlled comparison at three collaborating university sites included 242 patients diagnosed as having anxiety disorders. A two-week placebo washout period preceded random assignment to eight weeks of imipramine hydrochloride, chlordiazepoxide hydrochloride, or placebo treatment. Antianxiety effects of imipramine were superior to those of the other two agents by the second treatment week; these effects became more clearly significant thereafter and were independent of degree of both baseline depression and anxiety. Excluding patients with possible panic-phobic syndromes from the analyses removed most significant antiphobic and antidepressant effects of imipramine but left intact imipramine's significantly superior antianxiety effects.

Cognitive group psychotherapy of depression: the close-ended group.

One of the most important recent contributions to the understanding and treatment of depression is that of the cognitive theory and cognitive-behavior therapy developed by A. T. Beck. This paper describes the rationale and the technique of a short-term group psychotherapy based on Beck's Cognitive Therapy. The therapy was conducted with small closed-membership groups of patients with major depressive illness who were participating in a treatment-outcome study. Details of the role induction, session structure, roles of therapist and cotherapist, phases of group treatment course, and possible effectiveness are discussed. The technique presented here includes two individual cognitive sessions for purposes of role induction and establishing a therapeutic relationship before the first group session. We make use of flipcharts and a blackboard to illustrate various cognitive techniques, highly structured sessions with specific individualized agendas, defined therapist and cotherapist roles. The usual phases in a 15-session group course are described and our present experience of the techniques effectiveness are discussed.

The effects of stimulant medication on the growth of hyperkinetic children.

This article reviews the literature on possible growth-suppressing effects of stimulant medications in the long-term treatment of children with the hyperkinetic behavior syndrome. The evidence clearly indicates a temporary retardation in the rate of growth in weight and suggests a temporary slowing of growth in stature, but no effect on adult stature or weight. This temporary effect on growth is present during the first few years of treatment and seems related to drug dosage and to the presence or absence of drug holidays. These conclusions related specifically to treatment during the prepubertal period; little is known of the growth-related effects of treatment extending through pubescence.