Lack of inhibition by l-1-chloro-4-phenyl-3-toluene-p-sulphoamidobutan-2-one (l-1-tosylamido-2-phenylethyl chloromethyl ketone) of the formation of bacterial polypeptide chain initiation complex (Short Communication)

The chymotrypsin inhibitor l-1-chloro-4-phenyl-3-toluene-p-sulphonamidobutan-2-one does not inhibit the function of the initiation factor during the formation of the polypeptide chain initiation complex in vitro. Since the inhibitor has been shown previously to inhibit polypeptide chain elongation by reacting with elongation factor EF-Tu, the inhibitor can be used to investigate the initiation and elongation steps of protein biosynthesis separately.     

Competitive inhibition of Phe: tRNA ligase of sheep embryos by aurintricarboxylic acid]

Aurin tricarboxylic acid (A.T.A.), an inhibitor of protein biosynthesis (initiation and elongation steps), acts also as a competitive inhibitor of phenylalanine, in the ATP-PPi exchange and tRNAPhe aminoacylation reactions catalysed by cytoplasmic wheat germ phenylalanine:tRNA ligase.     

Occurrence of initiation factor 2 in the postribosomal fraction and identification of an initiation inhibitor as elongation factor G

The postribosomal fraction obtained at low salt concentrations from extracts of Escherichia coli contains substantial amounts of initiation factor 2. The apparent separation of initiation factor 2 into two distinct peaks upon chromatography on DEAE-cellulose results in part from an inhibitor that elutes within the initiation factor 2 peak. This inhibitor has been identified as elongation factor G. Elongation factor G possesses a ribosomal-dependent GTPase activity which reduces the amount of GTP available for initiation complex formation. This inhibitory effect can be overcome by high levels of GTP and a nucleotide triphosphate generating system.     

The intra-S-phase checkpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analyses

To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication-mediated DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. After a short dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in late-S-phase, compared to early-S-phase, cells and was due to both an inhibition of initiation and elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers. DNA replication was actively inhibited by checkpoint activation since 7-hydroxystaurosporine (UCN-01), the specific Chk1 inhibitor CHIR-124, or transfection with small interfering RNA targeting Chk1 restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced camptothecin-induced DNA damage at replication sites where histone γ-H2AX colocalized with replication foci. Together, our study demonstrates that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation.     

Regulation of translation elongation and phosphorylation of eEF2 in rat pancreatic acini

While pancreatic protein synthesis and the initiation of translation are regulated by hormones and neurotransmiters, whether the elongation process is also regulated is unknown. Stimulatory doses of cholecystokinin (CCK) (100 pM), bombesin (10 nM), and carbachol (10 microM) increased elongation rates (measured as ribosomal half-transit time) in pancreatic acini in vitro. At the same time these secretagogues reduced elongation factor 2 (eEF2) phosphorylation, the main factor known to regulate elongation, and increased the phosphorylation of the eEF2 kinase. The mTOR inhibitor rapamycin reversed the dephosphorylation of eEF2 induced by CCK, as did treatment with the p38 MAPK inhibitor SB202190, the MEK inhibitor PD98059, and the phosphatase inhibitor calyculin A. Neither rapamycin, SB202190, PD98059 nor calyculin A had an effect on CCK mediated eEF2 kinase phosphorylation. Translation elongation in pancreatic acinar cells is likely regulated by eEF2 through the mTOR, p38, and MEK pathways, and modulated through PP2A.     

Evidence for the presence of an inhibitor on ribosomes in mouse L cells infected with mengovirus.

After infection of mouse L cells with mengovirus, there is a rapid inhibition of protein synthesis, a concurrent disaggregation of polysomes, and an accumulation of 80S ribosomes. These 80S ribosomes could not be chased back into polysomes under an elongation block. The infected-cell 80S-ribosome fraction contained twice as much initiator methionyl-tRNA and mRNA as the analogous fraction from uninfected cells. Since the proportion of 80S ribosomes that were resistant to pronase digestion also increased after infection, these data suggest that the accumulated 80S ribosomes may be in the form of initiation complexes. The specific protein synthetic activity of polysomal ribosomes also decreased with time of infection. However, the transit times in mock-infected and infected cells remained the same. Cell-free translation systems from infected cells reflected the decreased protein synthetic activity of intact cells. The addition of reticulocyte initiation factors to such systems failed to relieve the inhibition. Fractionation of the infected-cell lysate revealed that the ribosomes were the predominant target affected. Washing the infected-cell ribosomes with 0.5 M KCI restored their translational activity. In turn, the salt wash from infected-cell ribosomes inhibited translation in lysates from mock-infected cells. The inhibitor in the ribosomal salt wash was temperature sensitive and micrococcal nuclease resistant. A model is proposed wherein virus infection activates (or induces the synthesis of) an inhibitor that binds to ribosomes and stops translation after the formation of the 80S-ribosome initiation complex but before elongation. The presence of such an inhibitor on ribosomes could prevent them from being remobilized into polysomes in the presence of an inhibitor of polypeptide elongation.     

Disruption of nuclear lamin organization blocks the elongation phase of DNA replication

The role of nuclear lamins in DNA replication is unclear. To address this, nuclei were assembled in Xenopus extracts containing AraC, a reversible inhibitor that blocks near the onset of the elongation phase of replication. Dominant-negative lamin mutants lacking their NH(2)-terminal domains were added to assembled nuclei to disrupt lamin organization. This prevented the resumption of DNA replication after the release of the AraC block. This inhibition of replication was not due to gross disruption of nuclear envelope structure and function. The organization of initiation factors was not altered by lamin disruption, and nuclei resumed replication when transferred to extracts treated with CIP, an inhibitor of the cyclin-dependent kinase (cdk) 2-dependent step of initiation. This suggests that alteration of lamin organization does not affect the initiation phase of DNA replication. Instead, we find that disruption of lamin organization inhibited chain elongation in a dose-dependent fashion. Furthermore, the established organization of two elongation factors, proliferating cell nuclear antigen, and replication factor complex, was disrupted by DeltaNLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.     

A relaxed discrimination of 2'-O-methyl-GTP relative to GTP between de novo and Elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B

Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2'-O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2'-O-methyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step ( approximately 150-fold) compared with approximately 2-fold at the elongation step. Interestingly, discrimination of the 2'-O-methyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the "flap," encompassing amino acids 443-454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2'-modified nucleotide analogues, because it is more permissive to inhibition than initiation.     

Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen

The use of small molecule inhibitors of cellular processes is a powerful approach to understanding gene function that complements the genetic approach. We have designed a high throughput screen to identify new inhibitors of eukaryotic protein synthesis. We used a bicistronic mRNA reporter to multiplex our assay and simultaneously screen for inhibitors of cap-dependent initiation, internal initiation and translation elongation/termination. Functional screening of >90 000 compounds in an in vitro translation reaction identified 36 inhibitors, 14 of which are known inhibitors of translation and 18 of which are nucleic acid-binding ligands. Our results indicate that intercalators constitute a large class of protein synthesis inhibitors. Four non-intercalating compounds were identified, three of which block elongation and one of which inhibits initiation. The novel inhibitor of initiation affects 5' end-mediated initiation, as well as translation initiated from picornaviral IRESs, but does not significantly affect internal initiation from the hepatitis C virus 5'-untranslated region. This compound should be useful for delineating differences in mechanism of initiation among IRESs.     

Synchronous replication of poliovirus RNA: initiation of negative-strand RNA synthesis requires the guanidine-inhibited activity of protein 2C.

We report that protein 2C, the putative nucleoside triphosphatase/helicase protein of poliovirus, is required for the initiation of negative-strand RNA synthesis. Preinitiation RNA replication complexes formed upon the translation of poliovirion RNA in HeLa S10 extracts containing 2 mM guanidine HCI, a reversible inhibitor of viral protein 2C. Upon incubation in reactions lacking guanidine, preinitiation RNA replication complexes synchronously initiated and elongated negative-strand RNA molecules, followed by the synchronous initiation and elongation of positive-strand RNA molecules. The immediate and exclusive synthesis of negative-strand RNA upon the removal of guanidine demonstrates that guanidine specifically blocks the initiation of negative-strand RNA synthesis. Readdition of guanidine HCl to reactions synchronously elongating nascent negative-strand RNA molecules did not prevent their continued elongation and completion. In fact, readdition of guanidine HCl to reactions containing preinitiation complexes elongating nascent negative-strand RNA molecules had no effect on subsequent positive-strand RNA synthesis initiation or elongation. Thus, the guanidine-inhibited function of viral protein 2C was not required for the elongation of negative-strand RNA molecules, the initiation of positive-strand RNA molecules, or the elongation of positive-strand RNA molecules. The guanidine-inhibited function of viral protein 2C is required only immediately before or during the initiation of negative-strand RNA synthesis. We suggest that guanidine may block an irreversible structural maturation of protein 2C and/or RNA replication complexes necessary for the initiation of RNA replication.     

Effects of novobiocin on adenovirus DNA synthesis and encapsidation.

Novobiocin, an inhibitor of DNA gyrase implicated in bacterial and likely mammalian, chromosome replication, inhibited the initiation, but not the elongation of human adenovirus DNA replicative synthesis. The inhibition was partially reversible, even in the presence of protein synthesis inhibitor. Novobiocin inhibited also the encapsidation of viral DNA, and this effect was independent of the block in DNA replication. It was suggested that novobiocin acted on two different functions, one involved in viral DNA replication initiation, the other in DNA encapsidation.     

Adenovirus cores can function as templates in in vitro DNA replication

Adenovirus cores prepared by gentle disruption of virus by heating at 56 degrees C in the presence of deoxycholate were able to function as templates in an in vitro DNA replication system, allowing both initiation, indicated by the formation of terminal protein-dCMP complex, and elongation of > 300 nucleotides. Using both cores and DNA-protein complexes as templates, it was also demonstrated that novobiocin, an inhibitor of DNA gyrase, inhibited in vitro DNA replication by preventing formation of the initiation complex.     

Role of the PI3K-TOR-S6K pathway in the onset of cell cycle elongation during Xenopus early embryogenesis

In the early embryogenesis of the frog, Xenopus laevis, cells proliferate by rapid and synchronous divisions, followed by cell cycle elongation and prolongation of the S phases, and then the appearance of the G2 and G1 phases after the midblastula transition (MBT). The beginning of cell cycle elongation was thought to depend on an increase in the nucleo-cytoplasmic (N/C) ratio in blastomeres and a decrease in cortical cytoplasmic factors necessary for cell cycle progression, although these factors are unknown. In the present study, we demonstrated that a regulatory subunit of PI3K (p85α) was localized in the cortical cytoplasm of the blastomere during the MBT. When the embryos were treated with a PI3K inhibitor, LY294002, or a TOR inhibitor, rapamycin, cell cycle elongation was initiated before the MBT. In addition, the inhibition of S6K expression by antisense morpholino oligo enhanced the initiation of cell cycle elongation. In contrast, the activation of PI3K-TOR by Rheb-S16H expression delayed the initiation of cell cycle elongation. These results indicate that a decrease in translational activity dependent on the PI3K-TOR-S6K pathway causes the initiation of cell cycle elongation at the onset of the MBT.     

Characterization of 2'(3')-trinitrophenyl-ATP as an inhibitor of ATP-dependent initiation complex formation between the DNA polymerase III holoenzyme and primed DNA

We have identified 2'(3')-trinitrophenyl-ATP to be an inhibitor of the ATP-dependent initiation complex formation reaction between the Escherichia coli DNA polymerase III holoenzyme and primed DNA. The inhibitor is specific for the initiation stage; once initiation complexes are formed the subsequent elongation reaction is unaffected. Three ATP-dependent DNA polymerase III holoenzyme reactions can be independently assayed: the ATP-dependent formation of initiation complexes, ATP binding, and the primed DNA-dependent hydrolysis of ATP. Trinitrophenyl ATP inhibits all three reactions to a similar extent with an apparent Ki between 6 and 15 microM in the presence of 5 microM ATP. This suggests all of these reactions are related and that they proceed through a common ATP-binding site. We include an improved purification of the DNA polymerase III holoenzyme in this report.     

eIF5A has a function in the elongation step of translation in yeast

The putative translation factor eIF5A is essential for cell viability and is highly conserved throughout evolution. Here, we describe genetic interactions between an eIF5A mutant and a translation initiation mutant (eIF4E) or a translation elongation mutant (eEF2). Polysome profile analysis of single and double mutants revealed that mutation in eIF5A reduces polysome run-off, contrarily to translation initiation mutants. Moreover, the polysome profile of an eIF5A mutant alone is very similar to that of a translation elongation mutant. Furthermore, depletion of eIF5A causes a significant decrease in total protein synthesis and an increase of the average ribosome transit time. Finally, we demonstrate that the formation of P bodies is inhibited in an eIF5A mutant, similarly to the effect of the translation elongation inhibitor cycloheximide. Taken together, these results not only reinforce a role for eIF5A in translation but also strongly support a function for eIF5A in the elongation step of protein synthesis.     

Initiation and elongation of protein synthesis: Their relative rates and sensitivities to inhibition in Chinese hamster ovary cells determined by a modified Edman procedure

The relative rates of initiation and elongation of protein synthesis are determined in exponentially growing cells from the incorporation of [³⁵S]methionine into N-terminal and internal positions of growing peptide chains by a modified Edman degradation. Sequential samples of labeled cells are precipitated into filter paper supports with trichloroacetic acid. Large groups of samples can then be incubated in bulk with phenylisothiocyanate. Individual samples are treated with trifluoroacetic acid, and the derivatized N-terminal amino acids extracted from the filter paper with ethylene dichloride. The percentage of incorporation which is N-terminal varies with the purity of the [³⁵S]methionine. Elevated temperature and hypertonicity, inhibitors of initiation, preferentially block incorporation into the N-terminal fraction (initiation) but allow continued incorporation into internal positions of previously initiated peptides. Puromycin inhibited incorporation into both fractions, as expected for an inhibitor of elongation.     

Inhibition of DNA-dependent RNA polymerase from Escherichia coli by pyran copolymer

Pyran copolymer, a potent inhibitor of DNA-dependent RNA polymerase from Escherichia coli, prevented polyribonucleotide synthesis by blocking both the initiation and elongation steps. The inhibition was noncompetitive with respect to template and nucleotide triphosphate substrates. Template binding and the stability of the nascent RNA chain were not affected by the inhibitor.     

Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae.

Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited.