Identification of inhibitors of bacterial transcription/translation machinery utilizing a miniaturized 1536-well format screen.

This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement. This multicomponent system permits identification of inhibitors at different steps in this pathway. Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives. The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff. Furthermore, the data demonstrate good agreement between IC(50) values derived for this assay in a 1536-well format and 384-well format.

Oxazolidinones mechanism of action: inhibition of the first peptide bond formation.

Oxazolidinones are potent inhibitors of bacterial protein biosynthesis. Previous studies have demonstrated that this new class of antimicrobial agent blocks translation by inhibiting initiation complex formation, while post-initiation translation by polysomes and poly(U)-dependent translation is not a target for these compounds. We found that oxazolidinones inhibit translation of natural mRNA templates but have no significant effect on poly(A)-dependent translation. Here we show that various oxazolidinones inhibit ribosomal peptidyltransferase activity in the simple reaction of 70 S ribosomes using initiator-tRNA or N-protected CCA-Phe as a P-site substrate and puromycin as an A-site substrate. Steady-state kinetic analysis shows that oxazolidinones display a competitive inhibition pattern with respect to both the P-site and A-site substrates. This is consistent with a rapid equilibrium, ordered mechanism of the peptidyltransferase reaction, wherein binding of the A-site substrate can occur only after complex formation between peptidyltransferase and the P-site substrate. We propose that oxazolidinones inhibit bacterial protein biosynthesis by interfering with the binding of initiator fMet-tRNA(i)(Met) to the ribosomal peptidyltransferase P-site, which is vacant only prior to the formation of the first peptide bond.

Mutational analysis of the hydrolytic activity of yeast RNA polymerase III.

For 25 mutant alleles of ret1, encoding the second largest subunit of yeast RNA polymerase III, we have studied the polymerase III nuclease activity, measuring both the total yield and dinucleotide product composition. Mutations affecting amino acids 309-325 gave slightly elevated nuclease activity. In region 367-376, two mutations gave 12-15-fold increased nuclease activity. Our results do not support the catalytic role in nuclease activity proposed for the conserved DDRD motif in this region (Shirai, T., and Go, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9056-9060). Mutations centered on a basic region from amino acids 480 to 490, which aligns with Escherichia coli beta-subunit sequences between Rif(r) clusters I and II, produce changes in the relative yields of A- and G-containing dinucleotides. Four such mutant polymerases pause during elongation at GPy sequences and, in addition, have a reduced frequency of termination at T(5) terminator sequences. We propose that the side chains of these mutationally altered amino acids are in direct contact with bases in the RNA-DNA hybrid very near the growing 3'-end. Two mutations in domain I near the C terminus produced very large increases in exonuclease activity and strongly increased termination, suggesting that this region also contacts the nascent RNA in the hybrid region.

Substrate specificity of the RNase activity of yeast RNA polymerase III.

Using yeast RNA polymerase III ternary complexes stalled at various positions on the template, we have analyzed the cleavage products that are retained and released by the transcription complexes. The retained 5' products result from cleavage at uridine residues during retraction, whereas the yield of mononucleotides and dinucleotides released indicates that multiple cuts occur near the 3' end. Comparison of the cleavage patterns of uridine-containing and 5-bromouridine-containing transcripts suggests that RNA within an RNA-DNA hybrid duplex is the substrate for the 3'-5' exonuclease. During transcription of the SUP4 tRNATyr gene, RNA polymerase III produces not only full-length pre-tRNATyr but also short oligonucleotides, indicating that exonuclease digestion and transcription are concurrent processes. To explore the possibility that these oligonucleotides are released by the action of the RNA polymerase III nuclease at previously observed uridine-rich pause sites, we tested modified templates lacking the arrest sites present in the SUP4 tRNATyr gene. Comparative studies of cleavage during transcription for these templates show a direct correlation between the number of natural pause sites and the yield of 3' products made. At the natural arrest sites and the terminator, RNA polymerase III carries out multiple cleavage resynthesis steps, producing short oligoribonucleotides with uridine residues at the 3' terminus.

In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.

We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467-1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA(Tyr) gene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.

Structure of the phenylalanyl-tRNA synthetase genes from Thermus thermophilus HB8 and their expression in Escherichia coli.

A 4459 bp long BamHI restriction fragment containing the two genes for the Thermus thermophilus HB8 phenylalanyl-tRNA synthetase was cloned in Escherichia coli and its nucleotide sequence was determined. The genes pheS and pheT encode the alpha- and beta-subunits with a molecular weight of 39 and 87 kD, respectively. Three conserved sequence motifs typical for class II tRNA synthetases occur in the alpha-subunit. Secondary structure predictions indicate that an arm composed of two anti-parallel alpha-helices similar to that reported for the E.coli seryl-tRNA synthetase may be present in its N-terminal portion. In the beta-subunit clusters of hydrophilic amino acids and a leucine zipper motif were identified, and several pronounced alpha-helical regions were predicted. The particular arginine and lysine residues in the N-terminal portion of the beta-subunit, which were found to participate in tRNA binding in the yeast and E.coli PheRSs, have their counterparts in the T.thermophilus protein. The 5'-portion of an open reading frame downstream of pheT was found and codes for a yet unidentified, extremely hydrophobic peptide. The pheST genes are presumably cotranscribed and translationally coupled. A novel type of a putative transcriptional terminator in Thermus species was identified immediately downstream of pheT and other Thermus genes. The genes pheS and pheST were expressed in E.coli.

A comparative study of the relationship between thermostability and function of phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophilus.

The relationship between thermostability and functional activities of phenylalanyl-tRNA synthetases (EC 6.1.1.20) from E. coli and Thermus thermophilus has been studied. In the case of the E. coli enzyme, the activity decreased after the 43 degrees C treatment, both in the [32P]PPi-ATP exchange reaction and the overall aminoacylation reaction, due to thermo-inactivation of the phenylalanyl-tRNA synthetase, whereas tRNA(Phe) preserved its native structure. In the Th. thermophilus system, the enzyme showed extreme thermostability (up to 90 degrees C), and the reduction in the tRNA aminoacylation rate after the 78 degrees C treatment was ascribed to denaturation of the tRNA(Phe). Since the enzyme did not lose the [32P]PPi-ATP exchange activity up to 85 degrees C, the observed lower thermo-resistance of the tRNA is evidence that the native structure of ribonucleic acids should be one of the most difficult to stabilize at high temperatures.

Comparative study of subunits of phenylalanyl-tRNA synthetase from Escherichia coli and Thermus thermophilus.

FPLC separation of alpha- and beta-subunits of phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8 has been carried out in the presence of urea. Native alpha-subunits of both enzymes were primarily alpha 2-dimers and tended to aggregate. Most E. coli enzyme beta-subunits were monomeric and only a small fraction was represented by beta 2-dimers. All thermophilic beta-subunits were beta 2-dimers. It was shown that monomers and all forms of homologous subunits had no catalytic activity in tRNA(Phe) aminoacylation. For the enzymes and their subunits, titration curves were obtained and isoelectric points were determined. The comparison of the relative surface charges indicated similarity of the surfaces of entire enzymes and the corresponding beta-subunits. Alpha-subunits displayed a distinctly different pH dependence of the surface charge. A spatial model of the oligomeric structure and a putative mechanism for its formation are discussed.

[A comparative study of phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophilus by the tritium topography method]. / Sravnitel'noe issledovanie fenilalanil-tRNK-sintetaz iz Escherichia coli i Thermus thermophilus metodom tritievoi planigrafii.

A comparative study of thermostability and amino acid composition of phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus HB8 has been carried out. In the thermophilic protein the proline, leucine, phenylalanine, arginine content was considerably increased, whereas that of asparagine, isoleucine, serine, threonine and lysine was decreased as compared to the mesophilic protein. Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of the both enzymes. In the E. coli enzyme the threonine residues were also easy to access, while on the surface of the thermophilic enzyme arginine residues were more abundant. A quantitative assay of the surface compositions revealed the increased exposure of (Leu + Ile) residues in the thermophilic protein as well as of the charged asparagine and arginine residues. A possible relationship of the observed effects to thermostability is discussed.

[Separation and comparative characteristics of subunits of phenylalanyl-tRNA synthetase from Escherichia coli MRE-600 and Thermus thermophilus HB8]. / Razdelenie i sravnitel'naia kharakteristika sub''edinits fenilalanil-tRNK-sintetaz iz Escherichia coli MRE-600 i Thermus thermophilus HB8.

Separation of alpha- and beta-subunits of phenylalanyl-tRNA-synthetases from E. coli MRE-600 and Thermus thermophilus HB8 using FPLC has been carried out for the first time. The separated subunits of both enzymes do not possess any detectable tRNA-amino-acylation activity. It was found that in the case of the E. coli enzyme beta-subunits exist in solution mainly in the monomeric form with negligible formation of beta 2-dimers, while alpha-subunits predominantly form alpha 2-dimers over the same concentration range. In the case of Thermus thermophilus phenylalanyl-tRNA-synthetase, both alpha- and beta-subunits mainly exist in the dimeric form. The putative mechanisms of alpha-subunit aggregation and of subunit association for alpha 2 beta 2-type enzymes are discussed.

Comparative study of the phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophlus by the tritium topography method.

A comparative study of thermostability and aminoacid composition of the phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus HB8 has been carried out. Compared with the mesophilic enzyme, a considerable increase of Pro, Leu, Phe, Arg and decrease of Asx, Ile, Ser, Thr and Lys content have been revealed in the thermophilic protein. Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of both the enzymes. In the E. coli enzyme, Thr residues were also easy to access while on the surface of the thermophilic enzyme there were more Arg residues. The quantitative assay of the surface compositions revealed the increased exposure of the (Leu + Ile) residues on the thermophilic protein as well as of the charged Asx and Arg residues. A possible correlation of the observed effects with thermostability is discussed.

[Activation of phagocytosis by preparations of human alpha and gamma interferons]. / Aktivatsiia fagotsitoza preparatami chelovecheskogo al'fa- i gamma-interferona.

Preparations of human alpha- and gamma-interferon differing by the method of production and purification had different capacity of activating phagocytosis of blood polymorphonuclear leukocytes from patients with dermatoses. Native preparations of alpha- and gamma-interferon had the highest phagocytosis-activating potency. A high level of this activity was also demonstrated with human leukocyte interferon II for injections purified by highly specific methods. A concentrated preparation of human leukocyte interferon for injections with a specific activity above 1 million IU/1 mg of protein and recombinant alpha 2-interferon (reaferon) exhibited minimal phagocytosis activating effect.

[Leukocyte migration inhibiting factor in preparations of alpha and gamma interferons]. / Faktor, ingibiruiushchii migratsiiu leikotsitov v preparatakh al'fa- i gamma-interferona.

High titres of leukocyte migration inhibition factor (LMIF) activity were found in native and partially purified preparations of human alpha- and gamma-interferon. In the course of chemical purification of interferon up to 98% of LMIF was lost. The highest content of LMIF was in gamma-interferon preparations, while recombinant alpha 2-interferon (reaferon) showed no LMIF activity.

[A macrophage migration-inhibiting factor in preparations of alpha- and gamma-interferon]. / Faktor, ingibiruiushchii migratsiiu makrofagov, v preparatakh al'fa- i gamma-interferona.

Native preparations of human alpha- and gamma-interferon synthesized in stationary, suspension or roller leukocyte cultures regularly showed high levels of migration inhibition factor (MIF) activity a significant portion of which was lost in the course of interferon purification and concentration. Minimal losses of MIF activity were achieved when medicinal preparations of interferon were made by the method of negative immunoabsorption on immobilized antibodies to inductor antigens and chromatography on Sephadex G-25.