Polypeptide Chain Elongation Research Articles

Growth phase-dependent transcription of the Streptomyces ramocissimus tuf1 gene occurs from two promoters.

The str operon of Streptomyces ramocissimus contains the genes for ribosomal proteins S12 (rpsL) and S7 (rpsG) and for the polypeptide chain elongation factors G (EF-G) (fus) and Tu (EF-Tu) (tuf). This kirromycin producer contains three tuf or tuf-like genes; tuf1 encodes the regular EF-Tu and is located immediately downstream of fus. In vivo and in vitro transcription analysis revealed a transcription start site directly upstream of S. ramocissimus tuf1, in addition to the operon promoter rpsLp. Transcription from these promoters appeared to be growth phase dependent, diminishing drastically upon entry into stationary phase and at the onset of production of the EF-Tu-targeted antibiotic kirromycin. In surface-grown cultures, a second round of tuf1 transcription, coinciding with aerial mycelium formation and kirromycin production, was observed. The tuf1-specific promoter (tuf1p) was located in the intercistronic region between fus and tuf1 by high-resolution S1 mapping, in vitro transcription, and in vivo promoter probing. During logarithmic growth, the tuf1p and rpsLp transcripts are present at comparable levels. In contrast to Escherichia coli, which has two almost identical tuf genes, the gram-positive S. ramocissimus contains only tuf1 for its regular EF-Tu. High levels of EF-Tu may therefore be achieved by the compensatory activity of tuf1p.

Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1α promoter and lac operator

We have constructed an inducible high-level expression vector, pEF-LAC. pEF-LAC has a modified human polypeptide chain elongation factor 1α (EF-1α) promoter containing three lactose operator sequences. Using the cat reporter gene, we characterized the transcriptional activity of pEF-LAC. In the transient transfection of NIH3T3 and BaF3 cells, the transcriptional activity of pEF-LAC was higher than that of the original human elongation factor 1α promoter, simian virus 40 (SV40) promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR). Cotransfection of the lactose repressor expression plasmid effectively suppressed the promoter activity of pEF-LAC, and the activity was fully recovered by addition of isopropyl β- d-thiogalactopyranoside (IPTG). Even in the stable transfection of Rat-1 cells, the promoter activity of the integrated pEF-LAC was much higher than that of the RSV-LTR and regulated in an IPTG-dependent manner. These results suggest that pEF-LAC is a useful vector for the inducible high-level expression of the cloned gene in a variety of mammalian cells.

Erythropoietin enhancer stimulates production of a recombinant protein by Chinese hamster ovary (CHO) cells under hypoxic condition.

Oxygen is a limiting nutrient in animal cell culture and its supply is still worthy of improvement for production of useful proteins with a high efficiency. From a different point of view, development of the system by which a high productivity can be maintained even under hypoxic condition as well as under normoxic condition may be important. A number of hypoxia-inducible genes have been found in eucaryotic cells and the induction in most cases, if not all, is due to hypoxic activation of the gene transcription. Transcription of erythropoietin gene is highly hypoxia-inducible and the induction is achieved by binding of a protein, which is widely distributed in animal cells, to a short DNA stretch (erythropoietin enhancer) in the 3' flanking region of erythropoietin gene. Using a hepatoma cell line (Hep3B) that produces the endogenous erythropoietin in an oxygen-dependent manner and Chinese hamster ovary cells that have been widely used for production of recombinant proteins, we show that, under hypoxic condition, the erythropoietin enhancer can activate not only the promoter of erythropoietin gene but also promoters of cytomegalovirus early genes and eucaryotic polypeptide chain elongation factor gene, both of which are very active in animal cells under normoxic condition.

Translational Activities of EF-Tu [G222D] Which Cannot Be Reconciled with the Classical Scheme of the Polypeptide Chain Elongation Cycle

We have developed a cell-free system ofE. colithat enables us to study thein vitrotranslation of natural mRNA mediated by wild-type or mutant EF-Tu. Various mutant EF-Tu species have been analyzed, one of which, EF-Tu [G222D], appeared to be virtually unable to mediate the translation of natural mRNA. Since this mutant factor is able to participate in translationin vivoby suppressing nonsense and frameshift mutations in cooperation with EF-Tu [A375T], a revision of the generally accepted scheme of the elongation cycle has been proposed (Bosch, L., Vijgenboom, E., & Zeef, L.A.H., 1996,Biochemistry36).

Mutation in the structural gene for release factor 1 (RF-1) of Salmonella typhimurium inhibits cell division.

A temperature-sensitive mutant of Salmonella typhimurium LT2 was isolated. At the nonpermissive temperature cell division stopped and multinucleated filaments were formed. DNA, RNA, or protein synthesis was not affected until after about two generations. Different physiological conditions, such as anaerobiosis and different growth media, suppress the division deficiency at high temperatures. Certain mutations causing a reduced polypeptide chain elongation rate also suppress the division deficiency. The mutation is recessive and shown to be in the structural gene for release factor I (prfA). DNA sequencing of both the wild-type (prfA+) and mutant (prfA101) allele revealed a GC-to-AT transition in codon 168. Like other known prfA mutants, prfA101 can suppress amber mutations. The division defect in the prfA101 mutant strain could not be suppressed by overexpression of the ftsQAZ operon. Moreover, at the nonpermissive temperature the mutant shows a normal heat shock and SOS response and has a normal ppGpp level. We conclude that the prfA101-mediated defect in cell division is not directed through any of these metabolic pathways, which are all known to affect cell division. We speculate that the altered release factor I induces aberrant synthesis of an unidentified protein(s) involved in the elaborate process of septation.

Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia

During normal spermatogenesis, more than half of the germ cells undergo apoptosis, but the physiological significance and molecular mechanisms of this programmed cell death are largely unknown. Because Bcl-2 functions as a death repressor, we have investigated the effect of misexpressing Bcl-2 in spermatogonia in transgenic mice using the human bcl-2 cDNA under the control of the human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter. In the 2-week-old transgenic testes, exogenous Bcl-2 was expressed in spermatogonia and massive accumulation of spermatogonia was observed in seminiferous tubules by 4 weeks. At this time, only a few spermatocytes were apparent, and the accumulated cells degenerated, leading to vacuolization in some seminiferous tubules by 7 weeks. In older transgenic mice, abnormal accumulation of spermatogonia and degeneration of these germ cells was still observed, but some seminiferous tubules in which the level of Bcl-2 expression was reduced recovered normal spermatogenesis. These observations indicate that spermatogonial apoptosis is part of the normal program of mammalian spermatogenesis and is regulated by a pathway affected by Bcl-2.

A revised bacterial polypeptide chain elongation cycle with a stepwise increase in restriction of unwanted ternary complexes by the ribosome.

A revised bacterial polypeptide chain elongation cycle with a stepwise increase in restriction of unwanted ternary complexes by the ribosome.

Isolation and characterisation of a recombinant, precursor form of lysosomal acid alpha-glucosidase.

Glycogenosis type II (GSD II, Pompe disease) is an autosomal recessive lysosomal storage disease that results from a deficiency of acid alpha-glucosidase (GAA). Patients with this disorder are unable to break down lysosomal glycogen, which consequently accumulates in the lysosome. To evaluate enzyme replacement therapy for GSD II patients, we have expressed human GAA cDNA in Chinese hamster ovary-K1 cells utilising a vector that places the cDNA under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line that secreted precursor recombinant GAA at approximately 18 mg.l-1.day-1 was identified. The precursor recombinant GAA was purified to homogeneity, had a molecular mass of 110 kDa as measured by SDS/PAGE, and was shown to have pH optima and kinetic parameters similar to those of GAA purified from human tissues. The partial N-terminal amino acid sequence of recombinant GAA conformed to that derived from the nucleotide sequence of the cloned cDNA. The recombinant enzyme was taken up by cultured fibroblasts and skeletal muscle cells from GSD II patients, and was shown to correct the storage phenotype. Endocytosed GAA was localised to the lysosome and showed evidence of intracellular processing to a more mature form. Activity levels increased up to twice the normal value and uptake was prevented if cells were cultured in the presence of mannose 6-phosphate.

1-Methylguanosine deficiency of tRNA influences cognate codon interaction and metabolism in Salmonella typhimurium

1-Methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in tRNA(1,2,3,Leu), tRNA(1,2,3,Pro), and tRNA(3Arg). A mutant of Salmonella typhimurium lacks m1G in these seven tRNAs when grown at or above 37 degrees C, as a result of a mutation (trmD3) in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. The m1G deficiency induced 24 and 26% reductions in the growth rate and polypeptide chain elongation rate, respectively, in morpholinepropanesulfonic acid (MOPS)-glucose minimal medium at 37 degrees C. The expression of the leuABCD operon is controlled by the rate with which tRNA(2Leu) and tRNA(3Leu) read four leucine codons in the leu-leader mRNA. Lack of m1G in these tRNAs did not influence the expression of this operon, suggesting that m1G did not influence the efficiency of tRNA(2,3Leu). Since the average step time of the m1G-deficient tRNAs was increased 3.3-fold, the results suggest that the impact of m1G in decoding cognate codons may be tRNA dependent. The trmD3 mutation rendered the cell more resistant or sensitive to several amino acid analogs. 3-Nitro-L-tyrosine (NT), to which the trmD3 mutant is sensitive, was shown to be transported by the tryptophan-specific permease, and mutations in this gene (mtr) render the cell resistant to NT. Since the trmD3 mutation did not affect the activity of the permease, some internal metabolic step(s), but not the uptake of the analog per se, is affected. We suggest that the trmD3-mediated NT sensitivity is by an abnormal translation of some mRNA(s) whose product(s) is involved in the metabolic reactions affected by the analog. Our results also suggest that tRNA modification may be a regulatory device for gene expression.

The tuf3 gene of Streptomyces coelicolor A3(2) encodes an inessential elongation factor Tu that is apparently subject to positive stringent control.

In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, respectively. While tuf1 appears to code for the major EF-Tu, the function of tuf3 is unknown. To assess the role of EF-Tu3, tuf3 was subjected to mutational and transcriptional analyses. Replacement of the 5'-half of tuf3 by an antibiotic resistance cassette had no detectable effect on phenotype, indicating that tuf3 is not essential for growth or differentiation. The transcription start site of tuf3 was located approximately 195 nt upstream of the translation start site. The sequence of the tuf3 promoter (Ptuf3) resembles the consensus for the major class of eubacterial promoters, and Ptuf3 was recognized preferentially by an RNA polymerase fraction enriched in sigma hrdB, the principal sigma factor of S. coelicolor. Nuclease S1 mapping failed to reveal tuf3 transcripts during growth of S. coelicolor in liquid culture, consistent with the apparent absence of EF-Tu3 in total protein extracts of the same strain. However, tuf3 transcription was observed in cultures of S. coelicolor M145 shortly after nutritional shiftdown (which resulted in the disappearance of tuf1 transcripts) and after addition of serine hydroxamate, both of which induce the stringent response. Transcription of tuf3 was also observed in transition-phase and stationary-phase cultures of S. coelicolor J1681, a strain deleted for bldA (which specifies a tRNA(Leu) for the rare leucine codon UUA). In all of these examples, transcription of tuf3 followed the production of ppGpp, consistent with the hypothesis that tuf3 is subject to positive stringent control.

The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA.

A Salmonella typhimurium LT2 mutant which harbors a mutation (miaB2508::Tn10dCm) that results in a reduction in the activities of the amber suppressors supF30 (tRNA(CUATyr)), supD10 (tRNA(CUASer)), and supJ60 (tRNA(CUALeu)) was isolated. The mutant was deficient in the methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A), a modified nucleoside that is normally present next to the anticodon (position 37) in tRNAs that read codons that start with uridine. Consequently, the mutant had i6A37 instead of ms2io6A37 in its tRNA. Only small amounts of io6A37 was found. We suggest that the synthesis of ms2io6A occurs in the following order: A-37-->i6A37-->ms2i6A37-->ms2io6A37. The mutation miaB2508::Tn10dCm was 60% linked to the nag gene (min 15) and 40% linked to the fur gene and is located counterclockwise from both of these genes. The growth rates of the mutant in four growth media did not significantly deviate from those of a wild-type strain. The polypeptide chain elongation rate was also unaffected in the mutant. However, the miaB2508::Tn10dCm mutation rendered the cell more resistant or sensitive, compared with a wild-type cell, to several amino acid analogs, suggesting that this mutation influences the regulation of several amino acid biosynthetic operons. The efficiencies of the aforementioned amber suppressors were decreased to as low as 16%, depending on the suppressor and the codon context monitored, demonstrating that the ms2 group of ms2io6A contributes to the decoding efficiency of tRNA. However, the major impact of the ms2io6 modification in the decoding process comes from the io6 group alone or from the combination of the ms2 and io6 groups, not from the ms2 group alone.

The beneficial effects of incubating bovine embryos in groups are due to platelet-derived growth factor

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, dP∗dt = M·f·n·At, where dP∗dt = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and At = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis (dP∗dt). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo−1 hr−1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo−1 hr−1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes (fzygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes (n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on fzygotic and dP∗dt, the rate of elongation of nascent histone polypeptide chains (At) during the first 6 hr of development was estimated; At remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.

Insulin-stimulated polypeptide chain elongation in the soleus muscle of mice.

The effect of insulin on polypeptide chain elongation was examined in soleus muscles isolated from 18 hour-fasted mice. Treatment with insulin for 1 hour increased the elongation rate, which was estimated by the half-transit time. This suggests that insulin stimulated protein synthesis by modifying the elongation rate in addition to the initiation rate.

Genetic Dissection of Synthesis and Function of Modified Nucleosides in Bacterial Transfer RNA

Publisher Summary This chapter discusses the transfer ribonucleic acid (tRNA) modification-deficient mutants that have been pivotal in unraveling their role in tRNA function. Unexpectedly, some modified nucleosides (m5U54, s4U8, and Q34), which occur frequently in tRNA, show no apparent functional impact on the tRNA based on the normal growth rate of the corresponding mutants. Other modified nucleosides (ms2ioGA37, mlG37, and W38, 39, 40) influence the function of the tRNA, and result in a reduced growth rate and polypeptide chain elongation rate for the mutant. Although, tRNA from all the three phylogenetic domains, archaea, bacteria, and eucarya, earlier called the kingdoms of archaebacteria, eubacteria, and eukaryotes, respectively contains modified nucleosides. A subset of these modified nucleosides is present in the tRNAs from all organisms. Moreover, some are even present in comparable positions in the tRNAs from all three domains. Therefore, the modified nucleosides may have been present in the tRNA of the progenitor. However, some modified nucleosides are domain-specific demonstrating that the presence of these modified nucleosides has evolved after the three phylogenetic domains had separated.

Characterization of the regulatory elements in the promoter of the human elongation factor-1 alpha gene.

Most eukaryotic cells abundantly express polypeptide chain elongation factor-1 alpha (EF-1 alpha), an enzyme which catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes. In this study, a series of deletion and scanning mutations was introduced in the promoter region of human EF-1 alpha chromosomal gene. Mutated promoters were fused to the bacterial chloramphenicol acetyltransferase gene, and their promoter activity was determined in human HeLa cells. These analyses indicated that both the 5'-flanking region and the first intron of the EF-1 alpha gene are essential for its promoter activity. The region responsible in the intron contains several Sp1 and Ap1 elements which seem to have additive effects on its promoter activity. In the 5'-flanking region, two cis-elements (EFP1 and EFP2) which work interdependently were identified. Gel shift assay with EFP1 and EFP2 elements indicated that several nuclear factors bind to EFP1 and EFP2, and one of the three retarded bands with EFP2 could be super-shifted with the anti-Sp1 antibody. These results indicate that Sp1 or its related factor cooperatively enhances the expression of the EF-1 alpha gene in the 5' flanking region.

The structural and functional basis for the kirromycin resistance of mutant EF-Tu species in Escherichia coli.

A structural and functional understanding of resistance to the antibiotic kirromycin in Escherichia coli has been sought in order to shed new light on the functioning of the bacterial elongation factor Tu (EF-Tu), in particular its ability to act as a molecular switch. The mutant EF-Tu species G316D, A375T, A375V and Q124K, isolated by M13mp phage-mediated targeted mutagenesis, were studied. In this order the mutant EF-Tu species showed increasing resistance to the antibiotic as measured by poly(U)-directed poly(Phe) synthesis and intrinsic GTPase activities. The K'd values for kirromycin binding to mutant EF-Tu.GTP and EF-Tu.GDP increased in the same order. All mutation sites cluster in the interface of domains 1 and 3 of EF-Tu.GTP, not in that of EF-Tu.GDP. Evidence is presented that kirromycin binds to this interface of wild-type EF-Tu.GTP, thereby jamming the conformational switch of EF-Tu upon GTP hydrolysis. We conclude that the mutations result in two separate mechanisms of resistance to kirromycin. The first inhibits access of the antibiotic to its binding site on EF-Tu.GTP. A second mechanism exists on the ribosome, when mutant EF-Tu species release kirromycin and polypeptide chain elongation continues.

Increased Expression of Acidic Ribosomal Protein (P0) mRNA After Phorbol Ester Treatment of Cultured Rat Thyroid (FRTL-5) Cells

P0, an acidic protein component of the ribosomal protein in eukaryotic 60 S ribosomal subunit, plays an important role in polypeptide chain elongation during translation. To investigate the role of protein kinase C in thyroid cell protein synthesis, we examined the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of P0 mRNA and protein. RNA slot blot hybridization revealed that TPA induced the accumulation of P0 mRNA in FRTL-5 cells in a time- and dose-dependent manner. A maximal increase of 2-fold was observed 18 h after addition of TPA. Cycloheximide markedly inhibited the TPA-induced accumulation of P0 mRNA. Nuclear runoff transcription assays using nuclei prepared from TPA-treated FRTL-5 cells revealed that TPA increased the transcription of P0 mRNA but not of β-actin. Immunoblotting experiments using anti-P protein antibody showed that TPA also increased the protein amount of P0. These results suggest that TPA activates protein synthesis in thyroid cells by inducing the expression of ribosomal proteins.

Stabilization of protein synthesis in thermotolerant cells during heat shock. Association of heat shock protein-72 with ribosomal subunits of polysomes.

Thermotolerance is defined as the capacity of cells, following a cycle of stress and recovery, to survive a second stress which would otherwise be lethal. Whereas this is a well-documented phenomenon, the mechanisms underlying this protective event remain to be elucidated. Protection of protein synthesis appears to be one of the components in the induction of thermotolerance termed "translational thermotolerance." In the present study we show that translational thermotolerance is not the result of an increase in the concentration of cellular transcripts or the stabilization of preexisting messages, nor the preservation of the rate of amino acid uptake, synthesis of aminoacyl-tRNA, or protection from degradation of newly synthesized polypeptides. These results suggest that translational thermotolerance is the consequence of stabilization of translational initiation and/or polypeptide chain elongation during heat shock. We found that heat shock protein (hsp)-72, the major inducible form of the hsp-70 family of heat shock proteins, is associated with ribosomal subunits in polysomes of thermotolerant cells during heat shock. We hypothesize that such interaction is responsible for rescuing translational initiation and/or polypeptide chain elongation in thermotolerant cells during a subsequent stress. It is possible that hsp-72 on the ribosome is "waiting" for the nascent polypeptide to emerge from the ribosome. Such interaction may maintain the growing polypeptide in solution during stress, allowing elongation to continue, and maintaining a constant rate of translation during the stress.

Soleus muscle nascent polypeptide chain elongation slows protein synthesis rate during non-weight-bearing activity

Protein synthesis rate of the soleus muscle decreases rapidly during non-weight-bearing activity. We isolated polysomes from 18-h non-weight-bearing soleus muscle to investigate the mechanism of this phenomenon. The distribution of polysomal alpha-actin mRNA and 18S rRNA on sucrose density gradients shows that polysomes shift to larger sizes (more ribosomes per mRNA) during non-weight-bearing activity. Furthermore, RNA is mobilized into the polysome pool of the non-weight-bearing soleus muscle; these data indicate that initiation of protein synthesis is not rate limiting. We explain these results as the slowing of nascent polypeptide chain elongation, such that there is a "traffic jam" of ribosomes on the mRNAs, increasing the number of ribosomes per mRNA while, at the same time, decreasing protein synthesis rate. In support of this hypothesis, myoblasts treated with a low dose of cycloheximide (a specific elongation inhibitor) show a similar shift in polysome size. A numerical model of protein synthesis further shows that elongation is more effective than initiation and termination in affecting protein synthesis and polysome size. We conclude that the non-weight-bearing-induced decrease in postural muscle protein synthesis rate is initially caused by slowing of nascent polypeptide chain elongation.

Peripheral blood mononuclear cells stimulated with C5a or lipopolysaccharide to synthesize equivalent levels of IL-1 beta mRNA show unequal IL-1 beta protein accumulation but similar polyribosome profiles.

Recent reports suggest that regulation of IL-1 beta gene expression in human monocytes may include translation level as well as transcription level control mechanisms. We studied IL-1 beta expression in PBMC stimulated with recombinant complement protein C5a or with LPS. IL-1 beta mRNA was expressed by C5a-treated PBMC, but very little or no IL-1 beta protein could be detected by ELISA or by Western blot analysis using a polyclonal Ab that reacts equally with pro-IL-1 beta and processed mature IL-1 beta. LPS-treated cells produced both IL-1 beta mRNA and protein. Velocity sedimentation analysis revealed, however, that IL-1 beta mRNA assembles into large polyribosomes in both C5a- and LPS-treated cells. Translation of the IL-1 beta mRNA in C5a-treated cells is not therefore blocked at the level of protein synthesis initiation. IL-1 beta mRNA was released from polyribosomes by treating the cells with puromycin, suggesting that the ribosomes are not frozen at the elongation phase of protein synthesis. One explanation for the data is that the rate of IL-1 beta polypeptide chain elongation or termination may be diminished in the C5a-stimulated PBMC.