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PubMed2004

Translation termination factors function outside of translation: yeast eRF1 interacts with myosin light chain, Mlc1p, to effect cytokinesis.

The translation termination factor eRF1 recognizes stop codons at the A site of the ribosome and induces peptidyl-tRNA hydrolysis at the peptidyl transferase centre. Recent data show that, besides translation, yeast eRF1 is also involved in cell cycle regulation. To clarify the mechanisms of non-translational functions of eRF1, we performed a genetic screen for its novel partner proteins. This screen revealed the gene for myosin light chain, Mlc1p, acting as a dosage suppressor of a temperature-sensitive mutation in the SUP45 gene encoding eRF1. eRF1 and Mlc1p are able to interact with each other and, similarly to depletion of Mlc1p, mutations in the SUP45 gene may affect cytokinesis. Immunofluorescent staining performed to determine localization of Mlc1p has shown that the sup45 mutation, which arrests cytokinesis, redistributed Mlc1p, causing its disappearance from the bud tip and the bud neck. The data obtained demonstrate that yeast eRF1 has an important non-translational function effecting cytokinesis via interaction with Mlc1p.

Cytokinesis↗

PubMed1989

Regulation of gene expression at the translational level. The rescue factor reverses thermosensitive protein synthesis in N4316, a conditionally-lethal mutant of Escherichia coli defective in translation.

Extracts of the conditionally-lethal mutant Escherichia coli N4316 are defective in a newly described translation factor, the rescue protein. We have analyzed the in vitro translation products of this mutant by gel electrophoresis during normal and arrested synthesis at the permissive and non-permissive temperatures. Translation programmed with MS2 bacteriophage RNA at the non-permissive temperature results in highly reduced synthesis of the coat protein with no detectable levels of the maturation and replicase products. Thus the relative number of copies of proteins synthesized by the ribosomes is altered in this mutant. In addition, there is mistranslation of the coat gene which results in the overproduction of the phage encoded no. 7 protein. Aberrant synthesis is also reflected in the increased read-through of termination codons during synthesis directed by phage RNAs harbouring amber mutations in the coat cistron. The rescue protein, purified from the parental strain, is able to complement the thermosensitive defect and restore proper synthesis. Biochemical characterization of the defect in the absence of rescue shows no detectable deficiency in the extent of initiation complex formation in reactions inhibited with sparsomycin. Peptidyltransferase is fully active as judged by the kinetics of formylmethionine-puromycin formation. However, rescue does exert an effect at the level of termination. In addition, the thermolability of the mutant can be reversed by dissociating 70S ribosomes into 30S and 50S subunits. Based on these and other observations, we propose tht rescue mediates a novel function in the association/dissociation of ribosomal subunits which is essential to the accuracy and efficiency of translation.

Bacterial Proteins↗

PubMed1985

In vitro translation of mRNA for arginine esterase, the major secretory protein of dog prostate, and in vitro processing of the translation product.

Poly(A)+ rich RNA was isolated from prostate of adult dogs and translated in the rabbit reticulocyte lysate cell-free protein-synthesizing system. Two-dimensional gel electrophoresis of the translation products showed that a protein with a molecular weight of 31 000 was predominantly synthesized. This protein was immunoprecipitated with antibodies directed against purified arginine esterase from dog seminal plasma. mRNA isolated from the prostate of animals castrated for 1 or 2 weeks was unable to direct the synthesis of arginine esterase. However, the synthesis of the enzyme could be stimulated by androgens in castrated animals, presumably by increasing prostatic concentrations of arginine esterase mRNA. The single chain translation product could be further processed in vitro by the addition of dog pancreas microsomes and purified arginine esterase. This procedure yielded split chains of arginine esterase which had identical electrophoretic mobilities as seminal plasma enzyme by two-dimensional gel electrophoresis. When prostatic tissue slices were incubated with tunicamycin, the unglycosylated arginine esterase obtained had a lower molecular weight than the in vitro translation product, suggesting that a signal peptide had been removed in the living cells. These results indicate that arginine esterase processing may include the following steps: removal of a signal peptide, glycosylation, and splitting of the polypeptide chain by active arginine esterase in the secretory granules or outside the cell.

Animals↗

PubMed1987

In vitro translation of human prostatic acid phosphatase mRNA and processing of the translation products by microsomal membranes and endoglycosidase H.

Poly(A)+ RNA was isolated from human prostatic tissue and translated in vitro in a rabbit reticulocyte lysate translation assay. Acid phosphatase labeled with [35S]methionine was immunoprecipitated with an antibody against seminal plasma acid phosphatase. Two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitate, followed by fluorography, revealed the presence of two spots (one major and one minor), both having a molecular mass of 43 kilodaltons (kDa) and an isoelectric point higher than mature acid phosphatase. Addition of canine pancreatic membranes to the translation assay resulted in the formation of four immunoprecipitable spots with molecular masses ranging from 43 to 49 kDa on one-dimensional gels. These spots probably represent acid phosphatases containing one to four core sugar groups, since after the addition of endoglycosidase H the molecular mass heterogeneity was abolished and we observed only one major band with a molecular mass (41 kDa) slightly lower than the ones of the primary translation product. These results suggest that human prostatic acid phosphatases are synthesized as two 43-kDa preproteins, which are further processed to 41-kDa proteins by removal of their signal peptide. Heterogeneity of the native protein arises mostly from glycosylation at four sites and not from differences in the amino acid sequence of the various forms.

Acetylglucosaminidase↗

PubMed2006

[Mnemonic strategy for english vocabulary of TCM fundamental theory formed by borrowing translation and morphemic translation from Western medical terms].

In the article, the importance and feasibility of borrowing translation and morphemic translation from Western medical terms for translating terms of TCM fundamental theory are briefly reviewed, and mnemonic strategy for the memorization of TCM vocabulary formed by the two translation methods is discussed.

Medicine, Chinese Traditional↗

PubMed2004

Translational and post-translational modifications of proteins as a new mechanism of action of alpha-interferon: review article.

Interferon-alpha (IFNalpha) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumours and melanoma. IFNalpha, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. However, the way by which tumour cell growth is directly suppressed by IFNalpha is not well known. Wide evidence exists on the possibility that cancer cells undergo apoptosis after the exposure to the cytokine. Here we will discuss data obtained by us and others on the post-translational regulation of the expression of proteins involved in the occurrence of apoptotic process such as tissue transglutaminase (tTG) or in the modulation of cell cycle such as the cyclin-dependent kinase inhibitor p27. This new way of regulation of p27 and tTG occurs through the modulation of their proteasome-dependent degradation induced by the cytokine. We will also review the involvement of protein synthesis machinery in the induction of cell growth inhibition by IFNalpha. In details, we will describe the effects of IFNalpha on the expression and activity of the protein kinase dependent from dsRNA (PKR) and on the eukaryotic initiation factor of protein synthesis 5A (eIF-5A) and their correlations with the regulation of cancer cell growth. These data strongly suggest that the antitumour activity of IFNalpha against human tumours could involve still unexplored mechanisms based on post-translational and translational control of the expression of proteins that regulate cell proliferation and apoptosis.

Animals↗

PubMed1998

Modulation of HPV16 E7 translation by tRNAs in eukaryotic cell-free translation systems.

Translational regulation of HPV16 E7 mRNA is of particular interest since the viral E7 protein has oncogenic activity. Here we report that additional supplementation of cell-free reticulocyte/wheat-germ translation systems with rat liver tRNA pool favors the expression of the viral oncoprotein E7 which otherwise is scarcely translatable. The translational activation is explained by the positive correlation between the frequency of the glutamic and aspartic codons in E7 mRNA, and the increased concentration of the corresponding tRNAs following tRNA supplementation. The present in vitro data suggest a link between E7 expression and cell conditions conductive to tRNA changes such as development, cell proliferation and aging.

Animals↗

PubMed1985

Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation.

The c, b and delta subunit genes of the Escherichia coli atp operon were cloned individually in an expression vector between the tac fusion promoter and the galK gene. The relative rates of subunit synthesis directed by the cloned genes were similar in vitro and in vivo and compared favourably with the subunit stoichiometry of the assembled proton-translocating ATP synthase of E. coli in vivo. The rate of synthesis of subunit c was at least six times that of subunit b and 18 times that of subunit delta. Progressive shortening of the long intercistronic sequence lying upstream of the subunit c gene showed that maximal expression of this gene is dependent upon the presence of a sequence stretching greater than 20 bp upstream of the Shine-Dalgarno site. This sequence thus acts to enhance the rate of translational initiation. The possibility that similar sequences might perform the same function in other operons of E. coli and bacteriophage lambda is also discussed. Translation of the subunit b cistron is partially coupled to translation of the preceding subunit c cistron. In conclusion, the expression of all the atp operon genes could be adjusted to accommodate the subunit requirements of ATP synthase assembly primarily by means of mechanisms which control the efficiency of translational initiation and re-initiation at the respective cistron start codons.

Base Sequence↗

PubMed1996

The influence of AUG codons in the hepatitis C virus 5' nontranslated region on translation and mapping of the translation initiation window.

The initiation of translation of hepatitis C virus (HCV) is cap-independent and mediated by an internal ribosome entry site (IRES) that is located in the 5' nontranslated region (5' NTR) of the viral genome. This 5' NTR is relatively long and folds into a complex structure involving multiple hairpins and a pseudoknot. Within the sequence encompassing the IRES there are several AUG triplets. Some of these AUG codons are conserved between HCV genotypes and the related pestiviruses. In this study the 5 AUG codons (positions 13, 32, 85, 96, and 215) that are present in the 5' NTR of the HCV H-strain have been mutagenized to determine their influence on HCV cap-independent translation. The effect of these mutations on the expression of a chloramphenicol acetyl transferase (CAT) gene was tested in vaccinia virus. vTF7-3 infected Hep2 cells transfected with plasmids for the expression of a monocistronic HCV 5' NTR-CAT mRNA. Mutating the AUG codons at positions 13, 32, and 215 does not have a significant effect on CAT expression, inactivating the AUG codons at either position 85 or position 96 severely impaired IRES function. To determine whether ribosomes scan the RNA to select the initiation site, AUG codons were inserted up- and downstream of the authentic HCV polyprotein translation initiation codon (position 342). Analysis of these mutants has revealed that the ribosome is unable to use an AUG codon that is placed either 7 nucleotides upstream or 8 nucleotides downstream of the inactivated AUG at position 342. These results indicate that when scanning is involved in the recognition of the translation initiating AUG, it is limited to a narrow region between nucleotides 335 and 350.

Chromosome Mapping↗

PubMed1995

Mevalonate regulates polysome distribution and blocks translation-dependent suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA: relationship to translational control.

We reported previously that 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis is regulated at the translational level by mevalonate. To determine at what stage mevalonate affects reductase synthesis, we examined the distribution of reductase mRNA in polysomes from cells treated with lovastatin alone; lovastatin and 25-hydroxycholesterol; or lovastatin, 25-hydroxycholesterol, and mevalonate. In lovastatin-treated cells, reductase mRNA was primarily associated with heavy polysome fractions. When 25-hydroxycholesterol was added to lovastatin-treated cells, reductase mRNA levels were reduced approximately fourfold in all polysome fractions, with no accompanying redistribution of reductase mRNA into lighter polysome fractions. However, addition of both 25-hydroxycholesterol and mevalonate to lovastatin-treated cells shifted reductase mRNA from heavier to lighter polysome fractions. No change in the distribution of control beta-actin or ribosomal protein S17 mRNA occurred with any of the treatments. These results suggest that mevalonate suppresses reductase synthesis at the level of initiation. When the translation inhibitor cycloheximide was added to all three regimens, reductase mRNA shifted into heavy polysome fractions. Treatment with either lovastatin alone or lovastatin plus 25-hydroxycholesterol resulted in a 50% greater loss of reductase mRNA from the heavy polysome fractions compared to the same fractions from noncycloheximide-treated cells. No loss of reductase mRNA occurred when cycloheximide was added to cells treated with both 25-hydroxycholesterol and mevalonate. beta-Actin mRNA levels and polysome distribution were not significantly changed by cycloheximide under any of these conditions. Translationally mediated suppression of reductase mRNA did not occur when protein synthesis was inhibited with puromycin. Our results indicate that regulation of reductase mRNA levels is translation-dependent and is linked to the rate of elongation.

Actins↗

PubMed2003

What's new in translation initiation? The first translation determines the fate of mRNA.

The two terms 'translation' and 'protein synthesis' are interchangeable in describing the process whereby the genetic code in the form of messenger RNA (mRNA) is deciphered such that amino acids cognate with the triplet code are joined end to end to form a peptide chain. However, new data suggest that the initial act of translation on newly synthesised mRNA also functions to proofread mRNA for errors. Aberrant mRNAs detected in this way are rapidly degraded before their encoded proteins impede normal cell function. Initiation of surveillance translation appears to differ from that of regular protein synthesis in three ways: (i) composition of the substrate; (ii) temporal and spatial restrictions; (iii) factors used to recruit the ribosome. This review discusses translational aspects of mRNA surveillance, primarily in the context of the mammalian system, although much information has come from studies in yeast and other organisms.

Animals↗

PubMed1998

Editing and translation of ribosomal protein S13 transcripts: unedited translation products are not detectable in maize mitochondria.

Maize mitochondrial transcripts for the ribosomal protein S13 gene (rps13) have six C- to -U editing sites, and each nucleotide conversion causes a change in the amino acid specified by the effected codons. Sequence analysis of 30 cDNA clones indicated that 73% of the cDNAS were edited at all six sites and 3% were completely unedited. Antibodies were produced against synthetic peptides that corresponded to unedited or edited translation products at editing sites V and VI (80% and 83% edited, respectively). Antibody preparations were purified that selectively recognized the edited or unedited forms of the epitope. The antibody preparations were highly sensitive to the amino-acid residue encoded at editing site VI, but relatively insensitive to the residue encoded at editing site V. Immunological analyses demonstrated that the edited translation product accumulated as a ribosomal protein, but that the unedited translation product was not detected in the mitochondrion, in the ribosomal fraction, or in a post-ribosomal supernatant. These results, taken together with other studies which demonstrated that incompletely edited transcripts are incorporated into polyribosomes, suggest that incompletely edited transcripts may be translated, but polypeptides encoded by incompletely edited RNAs may be unstable and, consequently, fail to accumulate.

Amino Acid Sequence↗

PubMed1979

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.

Animals↗

PubMed1989

Translational active mRNPs from rabbit reticulocytes are qualitatively different from free mRNA in their translatability in cell-free system.

The translatability of polyribosomal and free mRNPs from rabbit reticulocytes and their mRNA was compared. Both classes of mRNPs turned out to be active in rabbit reticulocyte lysates. Considerable differences between mRNPs and mRNA have been revealed. The most striking feature of mRNPs was that high concentrations of mRNPs do not inhibit protein biosynthesis, whereas high concentrations of mRNA strongly inhibit this process. This inhibition is specific for mRNA and does not occur at the addition of the same amount of rRNA from E. coli. The features of mRNP translation are not the result of addition of the supplementary translation factors within particles. The specific function of mRNP proteins in the process of translation is under discussion.

Animals↗

PubMed1989

Yeast, Saccharomyces cerevisiae, cell-free translation: the inhibition of translation by high temperature is reversible.

Yeast, Saccharomyces cerevisiae, extracts are inactive for translation at 37 degrees C. Two unexplained, simultaneously occurring phenomena appear to be responsible for this effect: (i) rapid inhibition of translation, and (ii) time-dependent inactivation of (a) translational component(s) at 37 degrees C. After short incubation of an extract at 37 degrees C, protein synthesis recovers efficiently after transfer of the extract to 23 degrees C. This behaviour of yeast cell-free systems enables the in vitro inactivation of temperature-sensitive translational components and therefore facilitates studies with extracts derived from temperature-sensitive strains.

Electrophoresis, Polyacrylamide Gel↗

PubMed1991

Potassium salts influence the fidelity of mRNA translation initiation in rabbit reticulocyte lysates: unique features of encephalomyocarditis virus RNA translation.

It is widely assumed that in vitro translation of mRNA is more efficient in the presence of potassium acetate rather than KCl, that the optimum concentration of potassium acetate is higher than for KCl, and that uncapped RNAs exhibit a lower optimum salt concentration than capped mRNAs. When these assumptions were examined using several different mRNA species in four batches of rabbit reticulocyte lysate, some notable exceptions were found. The translation of encephalomyocarditis virus (EMCV) RNA exhibited a salt optimum unusually high for an uncapped mRNA, and was very much more efficient and accurate with KCl rather than potassium acetate. It was also unique in being strongly activated by low concentrations (5-10 mM) KSCN in the presence of 90 mM potassium acetate. For the translation of other uncapped RNAs (poliovirus RNA, cowpea mosaic virus (CPMV) M RNA and bacteriophage MS2 RNA) amino acid incorporation at the optimum potassium acetate level was significantly greater than could be achieved using KCl. However, KCl was found to be restrictive and potassium acetate permissive for the synthesis of abnormal products thought to arise from initiation at incorrect sites, with the result that KCl gave a product pattern closer to that observed in vivo. In the particular case of the reticulocyte lysate system, accurate translation therefore requires the use of KCl rather than potassium acetate, but the choice of salt was found to be less critical in cell-free extracts from HeLa or L-cells.

Animals↗

PubMed1992

Regulation of transcription by translational components in coupled translation-transcription cell-free system.

A coupled translation-transcription cell-free system was established from eukaryotic cells. The biosynthetic activity of this coupled system closely resembles the synthetic behavior of cells in vivo, and exhibits regulatory phenomena similar to that of intact cells. The translational system consists of rabbit reticulocyte lysate, or its components fractionated by centrifugation. The transcriptional portion consists of cockerel liver nuclei. Incorporation of amino acids into protein by the coupled system is linear for hours. Similarly, transcription in the coupled system is continuous for hours and is proportional with time. More than 90% of the transcriptional products are secreted into the incubation medium. The components of the translational system influence and regulate transcriptional activities. In the presence of ribosomes the nuclei transcribe mostly poly(A)+ RNA with alpha-amanitin sensitivity consistent with activation of RNA polymerase II. Hybrid selection experiments demonstrate authentic preproalbumin mRNA among the transcriptional products. The putative mRNA secreted into the medium in the coupled system is found on polysomes, indicating translation of de novo synthesized message. Addition of excess reticulocyte mRNP to the medium of the coupled system results in transcription of primarily ribosomal RNA, 5S RNA, and tRNA, the products of RNA polymerases I and III. These activities closely imitate the behavior of liver in vivo under conditions of nutritional shifts or hormonal influences. The coupled system transcribes, processes, and transports substantial quantities of RNA, about 1.6 micrograms/10(6) nuclei/h. Thus, a coupled system has been established that lends itself to the exploration of regulatory interactions of cell components as it appears to closely resemble the in vivo situation.

Animals↗

PubMed1999

Translational suppressors and antisuppressors alter the efficiency of the Ty1 programmed translational frameshift.

Certain viruses, transposons, and cellular genes have evolved specific sequences that induce high levels of specific translational errors. Such "programmed misreading" can result in levels of frameshifting or nonsense codon readthrough that are up to 1,000-fold higher than normal. Here we determine how a number of mutations in yeast affect the programmed misreading used by the yeast Ty retrotransposons. These mutations have previously been shown to affect the general accuracy of translational termination. We find that among four nonsense suppressor ribosomal mutations tested, one (a ribosomal protein mutation) enhanced the efficiency of the Tyl frameshifting, another (an rRNA mutation) reduced frameshifting, and two others (another ribosomal protein mutation and another rRNA mutation) had no effect. Three antisuppressor rRNA mutations all reduced Tyl frameshifting; however the antisuppressor mutation in the ribosomal protein did not show any effect. Among nonribosomal mutations, the allosuppressor protein phosphatase mutation enhanced Tyl frameshifting, whereas the partially inactive prion form of the release factor eRF3 caused a slight decrease, if any effect. A mutant form of the other release factor, eRF1, also had no effect on frameshifting. Our data suggest that Ty frameshifting is under the control of the cellular translational machinery. Surprisingly we find that translational suppressors can affect Ty frameshifting in either direction, whereas antisuppressors have either no effect or cause a decrease.

Base Sequence↗