We present a next-to-leading order calculation for the virtual photoproduction of one and two jets in ep collisions. Soft and collinear singularities are extracted using the phase space slicing method. The collinear photon initial state singularity depends logarithmically on the mass of the virtual photon and is absorbed into the virtual photon structure function. An MS factorization scheme is defined similarly to the real photon case. Numerical results are presented for HERA conditions using the Snowmass jet definition for inclusive single jet and dijet cross sections. We study the dependence of these cross sections on the transverse energies and rapidities of the jets. Finally, we compare the ratio of the experimentally defined resolved and direct cross sections with recent ZEUS data as a function of the photon virtuality P 2 .

The three ribonucleic acids (RNAs) from Escherichia coli ribosomes were isolated and then labeled at their 3' ends by oxidation with periodate followed by reaction with thiosemicarbazides of fluorescein or eosin. Ribosomal subunits reconstituted with the labeled RNAs were active for polyphenylalanine synthesis. The distances between the 3' ends of the RNAs in 70S ribosomes were estimated by nonradiative energy transfer from fluorescein to eosin. The percentage of energy transfer was calculated from the decrease in fluorescence lifetime of fluorescein in the quenched sample compared to the unquenched sample. Fluorescence lifetime was measured in real time by using a mode-locked laser for excitation and a high-speed electrostatic photomultiplier tube for detection of fluorescence. The distances between fluorophores attached to the 3' ends of 16S RNA and 5S RNA or 23S RNA were estimated to be about 55 and 71 A, respectively. The corresponding distance between the 5S RNA and 23S RNA was too large to be measured reliably with the available probes but was estimated to be greater than 65 A. Comparison of the quantum yields of the labeled RNAs free in solution and reconstituted into ribosomal subunits suggests that the 3' end of 16S RNA does not interact appreciably with other ribosomal components and may be in a relatively exposed position, whereas the 3' ends of the 5S RNA and 23S RNA may be buried in the 70S ribosomal subunit.

Large subunits of E. coli ribosomes, specifically 23S rRNA, have the capacity to mediate refolding of denatured rhodanese. Refolding activity is related to the state or conformation of ribosomes that is promoted by EF-G. Activation by either mechanism is strongly inhibited by the EF-G.GDP.fusidic acid complex.

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Highly purified preparations of the heme-controlled eIF-2 alpha (eukaryotic peptide initiation factor 2 alpha subunit) kinase of rabbit reticulocytes contain an abundant 90-kilodalton (kDa) peptide that is immunologically cross-reactive with spectrin and that modulates the activity of the enzyme [Kudlicki, W., Fullilove, S., Read, R., Kramer, G., & Hardesty, B. (1987) J. Biol. Chem. 262, 9695-9701]. The amino-terminal sequence of the 90-kDa protein has a high degree of similarity with the known amino-terminal sequences of the Drosophila 83-kDa heat shock protein (20 out of 22 residues) and with other related heat shock proteins. The amino acid sequence of a tryptic phosphopeptide isolated by high-performance liquid chromatography from the eIF-2 alpha kinase associated 90-kDa protein after phosphorylation by casein kinase II is shown to be identical with a 14 amino acid segment of the known sequence of the Drosophila 83-kDa heat shock protein. Results of hydrodynamic studies indicate a highly elongated structure for the reticulocyte protein, characteristic of a structural protein. Additional structural similarities between the eukaryotic heat shock proteins, the reticulocyte eIF-2 alpha kinase associated 90-kDa peptide, and spectrin are discussed.

One of the most ancient tRNA modifications, present in all organisms as well as mitochondria and chloroplasts, is methylation of the N1 atom of guanosine at position 37 (m 1 G37) 3 (1). The m 1 G37 modification is catalyzed by a tRNA (guanine-N1-)-methyltransferase (EC 2.1.1.31) encoded by trmD in bacteria or TRM5 in archaea and eukaryotes (1-3). Remarkably, the bacterial trmD gene is not homologous to TRM5; thus, the m 1 G37-modifying enzyme evolved twice. Trm5p has been shown to be responsible for m 1 G37 methylation of at least eight cytoplasmic tRNAs in Saccharomyces cerevisiae (1, 4). trm5 mutants that lack this modification exhibit a severe growth defect (1), consistent with the important role of m 1 G37 methylation in reading frame maintenance (5).S. cerevisiae also has at least eight mitochondrially encoded tRNAs that carry the m 1 G37 modification (6), including the initiator tRNA (tRNA Met f ) (7) and tRNA Phe (8). The enzyme(s) responsible for modifying these mitochondrial tRNAs has not been identified. There is no apparent homolog of bacterial trmD in eukaryotic or mitochondrial genomes, so it has been proposed (1, 9) that Trm5p might also be responsible for methylation of mitochondrial tRNAs. To date, however, only cytoplasmic and nuclear localization of Trm5p has been reported (10). Therefore, it is possible that yeast encode a separate mitochondrial m 1 G37 methyltransferase enzyme. To address this question, we have cloned, purified, and characterized the S. cerevisiae TRM5-encoded protein. We show that it possesses tRNA methyltransferase activity on both natural and synthetic mitochondrial tRNA substrates and is specific for methylation of the N1 atom of guanosine at position 37. Furthermore, we show that Trm5p is localized to both the cytoplasm and mitochondria in yeast. Thus, the protein encoded by the TRM5 gene in yeast represents yet another example of dual protein localization from a single gene (11,12). EXPERIMENTAL PROCEDURESChemicals, Reagents, and Strains-Reagent grade chemicals were purchased from Sigma, Fisher, or VWR and used without further purification. Geneticin (G-418 sulfate) was obtained from American Bioanalytical. SUPERase-in, RPA III ribonuclease protection assay kit, and RNA marker templates were from Ambion. The commercially produced enzymes (and their distributors) were ribonuclease P 1 (Sigma), phosphodiesterase I * This work was supported in part by Robert A. Welch Foundation Grant F-1576 (to D. E. G.) and the U. S. Army Research Office Grant . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The translation elongation factor (EF) Tu has chaperone-like capacity to promote renaturation of denatured rhodanese. This renaturation activity is greatly increased under conditions in which the factor can oscillate between the open and closed conformations that are induced by GDP and GTP, respectively. Oscillation occurs during GTP hydrolysis and subsequent replacement of GDP by EF-Ts which is then displaced by GTP. Renaturation of rhodanese and GTP hydrolysis by EF-Tu are greatly enhanced by the guanine nucleotide exchange factor EF-Ts. However, renaturation is reduced under conditions that stabilize EF-Tu in either the open or closed conformation. Both GDP and the nonhydrolyzable analog of GTP, GMP-PCP, inhibit renaturation. Kirromycin and pulvomycin, antibiotics that specifically bind to EF-Tu and inhibit its activity in peptide elongation, also strongly inhibit EF-Tu-mediated renaturation of denatured rhodanese to levels near those observed for spontaneous, unassisted refolding. Kirromycin locks EF-Tu in the open conformation in the presence of either GTP or GDP, whereas pulvomycin locks the factor in the closed conformation. The results lead to the conclusion that flexing of EF-Tu, especially as occurs between its open and closed conformations, is a major factor in its chaperone-like refolding activity.

Highly purified preparations of hemin-controlled repressor of rabbit reticulocyte contain a 3':5'-cyclic AMP-independent protein kinase activity that phos horylates the low-molecular-weight (about 38,000) Globin synthesis in both rabbit reticulocytes (1, 2) and their cell-free lysates (3, 4) is controlled by the availability of hemin. The cessation of protein synthesis observed in the absence of added hemin is due to a block in initiation of new globin chains (3-7) and appears to involve the depletion of 40S ribosomal subunit-Met-tRNAf initiation complexes (8,9). An inhibitory protein, the hemin-controlled repressor (HCR), has been isolated from reticulocyte lysates incubated without hemin (10) which, when added to hemin-supplemented lysates, produced inhibition kinetics similar to those induced by hemin deficiency (11) and promotes the disappearance of 40S-Met-tRNAf initiation complexes (8,12). Hunt and coworkers (13,14) have reported that millimolar amounts of 3':5'-cyclic AMP (cAMP), 2-aminopurine, or GTP prevent the inhibition of protein synthesis observed in reticulocyte lysates incubated in the absence of hemin. They also observed that ATP counteracted the effect of GTP (14) In this communication we report that highly purified HCR preparations, while devoid of histone kinase activity, contain a cAMP-independent protein kinase activity that phosphorylates the 38,000-dalton polypeptide chain of IF-E2 and certain proteins associated with reticulocyte 408 ribosomal subunits that have been washed with 0.5 M KC1. Furthermore, goat IgG antibody against HCR neutralizes the above-mentioned kinase activity as well as the inhibitory activity of HCR while not having an effect on the histone kinase activity. METHODSFractionation of Reticulocyte and Artemia safina Components. Preparation of rabbit reticulocyte ribosomes has been described (18). A. salina ribosomes and their subunits were prepared by the procedure of Zasloff and Ochoa (19) as modified by Kramer et al. (20). Subunits of reticulocyte ribosomes were prepared by the procedure of Falvey and Staehelin (21) as modified by Obrig et al. (22). Ribosomal subunits, concentrated by centrifugation, were active in protein synthesis.The hemin-controlled repressor, HCR, from rabbit reticulocytes was purified essentially by the procedure of Gross and Rabinovitz (10) except that the order of the steps involved was altered. The postribosomal supernatant fraction of a 1:1 reticulocyte/water lysate was made 5 mM in N-ethylmaleimide, incubated for 15 min at 370, then made 5 mM in dithioerythritol and incubated at 370 for 10 min. The treated supernatant was then precipitated with ammonium sulfate between the levels of 0 and 50% saturation; the precipitate was fractionated by successive chromatography on DEAE-cellulose, hydroxylapatite, phosphocellulose, DEAE-cellulose, and Sepharose 6B. The resulting HCR preparation gives 50% inhibition of protein synthesis in a reticulocyte lysate at a concentration of 0.4 gg/ml. The 0.5 M KC1 ribosomal wash fraction of rabbit reticu...