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At least 307 records · Page 17Linked to original sources

PubMed1997

Translation of the mRNA for the sporulation gene spoIIID of Bacillus subtilis is dependent upon translation of a small upstream open reading frame.

We report the existence of a small open reading frame (usd) that is located between the promoter and coding sequence for the sporulation gene spoIIID in Bacillus subtilis. The mRNA from the usd-spoIIID operon contains an inverted repeat sequence that is predicted to form a stem-loop structure that would sequester the ribosome binding site for spoIIID. A mutation eliminating the ribosome binding site for the upstream open reading frame caused an oligosporogenous phenotype and interfered with the translation, but not the transcription, of the downstream gene spoIIID. We propose that efficient synthesis of SpoIIID requires that the putative stem-loop structure be disrupted by translation through the upstream open reading frame.

Amino Acid Sequence↗

PubMed1989

Pre-translational and post-translational regulation of TSH synthesis in normal and neoplastic thyrotrophs.

We are interested in the mechanisms by which endocrine and developmental factors regulate TSH synthesis at both pre-translational and post-translational levels. Thyroid hormone profoundly decreases transcription of the TSH-beta gene, while TRH and agents modifying cyclic AMP increase transcription. To elucidate the molecular mechanisms underlying these effects, human embryonal kidney cells were transfected with constructs of the human TSH-beta gene fused to the chloramphenicol acetyltransferase gene. The first exon of human TSH-beta, contains an element that increases basal expression and mediates T3-induced gene repression, probably through a direct interaction with c-erbA beta. This transcriptional repression by T3 appears aberrant in thyrotropic tumors. In contrast, TRH and agents modifying cyclic AMP mediate increased transcription of TSH-beta through interacting with upstream regulatory elements. Thyroid hormone, TRH and developmental factors also regulate the branching pattern and relative sialylation of TSH carbohydrate chains, which may affect TSH action in vitro and in vivo. Certain thyrotropic tumors produce TSH with more complex carbohydrate branching patterns, which may increase its biologic activity.

Humans↗

PubMed2005

A Graves' disease-associated Kozak sequence single-nucleotide polymorphism enhances the efficiency of CD40 gene translation: a case for translational pathophysiology.

We analyzed the mechanism by which a Graves' disease-associated C/T polymorphism in the Kozak sequence of CD40 affects CD40 expression. CD40 expression levels on B cells in individuals with CT and TT genotypes were decreased by 13.3 and 39.4%, respectively, compared with the levels in CC genotypes (P = 0.012). Similarly, Rat-2 fibroblasts transfected with T-allele cDNA expressed 32.2% less CD40 compared with their C-allele-transfected counterparts (P = 0.004). Additionally, an in vitro transcription/translation system showed that the T-allele makes 15.5% less CD40 than the C-allele (P < 0.001), demonstrating that the effect of the single-nucleotide polymorphism (SNP) on CD40 expression is at the level of translation. However, the SNP did not affect transcription, because the mRNA levels of CD40, as measured by quantitative RT-PCR, were independent of genotype. Therefore, our results may suggest that the C allele of the CD40 Kozak SNP, which is associated with Graves' disease, could predispose to disease by increasing the efficiency of translation of CD40 mRNA.

5' Untranslated Regions↗

PubMed1997

The Mutation of mRNA Translation Initiation Region Influences Gene Translation.

In order to investigate the relationship between the structure of the mRNA translation initiation region (TIR) and gene expression, We mutated multiple sites of the 5' end of IFN-alpha8 and GM-CSF genes by site-directed mutagenesis without changing their amino acid sequences. SDS-PAGE showed that the protein products of mutated genes increased greatly in recombinant clones, as compared with their native genes. RNA dot blot revealed that the difference of their corresponding amount of mRNA transcribed between the native and the mutated genes was negligible. These results imply that the elevated expressions are attributed mainly to increased translation level. The prediction of mRNA secondary structure suggests that the delta G of TIR may have close relations to the expression level.

Journal Article↗

PubMedDate not supplied

[Influence of individual domains of the translation termination factor eRF1 on induction of the GTPase activity of the translation termination factor eRF3].

Translation termination in eukaryotes is governed by two proteins, belonging to the class-1 (eRF1) and class-2 (eRF3) polypeptide release factors. eRF3 catalyzes hydrolysis of GTP to GDP and inorganic phosphate in the ribosome in the absence of mRNA, tRNA, aminoacyl-tRNA and peptidyl-tRNA but needs the presence of eRF1. It's known that eRF1 and eRF3 interact with each other in vitro and in vivo via their C-terminal regions. eRF1 consists of three domains - N, M, and C. In this study we examined the influence of individual domains of the human eRF1 on induction of the human eRF3 GTPase activity in the ribosome in vitro. It was shown that none of the N-, M-, C- and NM-domains induces eRF3 GTPase activity in presence of the ribosomes. MC-domain does induce GTPase activity of eRF3 but four times less efficient than full-length eRF1, therefore, MC-domain (and very likely M-domain) binds to the ribosome in the presence of eRF3. Based on these data and taking into account the data available in literature, a conclusion was drawn that the N domain of eRF1 is not essential for eRF1-dependent induction of the eRF3 GTPase activity. A working hypothesis is formulated, postulating that GTPase activity eRF3 during the translation termination is associated with the intermolecular interactions of GTP/GDP, GTPase center of the large ribosomal subunit (60S), MC-domain of eRF1, C-terminal region and GTP-binding domains of eRF3, but without participation of the N-terminal region of eRF3.

Animals↗

PubMed1981

Processing in vivo of precursor maltose-binding protein in Escherichia coli occurs post-translationally as well as co-translationally.

The mechanism of synthesis of maltose-binding protein (Mr = 38,500), an exported periplasmic protein in Escherichia coli, was investigated in vivo. A precursor to maltose-binding protein (Mr - 41,000), which is identical to the precursor polypeptide synthesized in vitro in a cell-free system, can be detected in vivo indicating that it is not processed to mature size until the polypeptide chain is terminated. The population of incomplete, nascent polypeptide chains of maltose-binding protein was found to contain NH2 termini characteristic of both precursor and mature protein demonstrating that processing occurs co-translationally as well as post-translationally. However, the polypeptide containing the signal sequence must reach a critical size of Mr - 33,000 before any processing takes place.

ATP-Binding Cassette Transporters↗

PubMed1989

In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors.

Surfactant proteolipid SP-B is a hydrophobic protein of Mr = 8000 identified in organic solvent extracts of pulmonary surfactant. Analysis of the human SP-B RNA predicts that the active surfactant peptide is derived by proteolysis of an Mr = 40,000 precursor. In the present work, characteristics of synthesis, secretion and processing of SP-B were demonstrated in a pulmonary adenocarcinoma cell line by immunoprecipitation of radiolabelled precursors. Treatment of cells with tunicamycin resulted in synthesis and secretion of unglycosylated proSP-B of Mr = 39,000. Immunoprecipitation of protein produced by in vitro translation of human lung poly(A)+ RNA detected an Mr = 40,000 protein; the size discrepancy is likely related to cleavage of a leader signal sequence. Endoglycosidase-H-sensitive precursors of Mr = 41,000-43,000, pI = 5.1-5.4 were the first isoforms detected within the cells and were processed to endoglycosidase-H-resistant isoforms and secreted. Neuraminidase and endoglycosidase-F-sensitive forms of proSP-B were first detected in the media at 60 min as Mr = 42-46,000 isoforms with pI = 4.6-5.1. Proteolytically processed isoforms of proSP-B were detected primarily in the media and were generated by cleavage of an amino-terminal Mr = 16,000 peptide resulting in Mr = 27,000-33,000 isoforms (pH = 5.6-6.8). The Mr = 27,000-33,000 isoforms were sensitive to neuraminidase, resulting in isoforms with pH = 6.0-6.8. Digestion of the Mr = 27,000-33,000 peptide with endoglycosidase-F resulted in isoforms of Mr = 23,000, pH = 6.0-6.8. The endoglycosidase-F-resistant peptide of Mr = 16,000, pI = 4.2-4.4 was identified with an antiserum generated against synthetic peptides derived from the amino-terminal domain, as deduced from the SP-B DNA sequence. Further proteolytic processing of the Mr = 27,000-33,000 isoforms to the Mr = 8000 peptide detected in surfactant was not observed in this cell line. Thus, in the H441-4 cells (a cell line with morphologic features of Clara cells), SP-B is synthesized as a preproprotein which undergoes cleavage of a signal sequence and addition of asparagine-linked carbohydrate; proSP-B is secreted by processes which are independent of glycosylation. SP-B peptides of Mr = 27,000-33,000 and Mr = 16,000, representing carboxy and amino-terminal domains, accumulate in the media.

Electrophoresis, Polyacrylamide Gel↗

PubMed1997

Multiplicity of the beta form of the cAMP-dependent protein kinase inhibitor protein generated by post-translational modification and alternate translational initiation.

Two distinct species of the thermostable inhibitor of the cAMP-dependent protein kinase, PKIalpha and PKIbeta, exist that are the products of separate genes. The PKIbeta form, as first isolated from rat testis, is a 70-amino acid protein, but the genomic sequence suggested that an alternate form might exist, arising as a consequence of alternate translational initiation. This species, now termed PKIbeta-78, has been synthesized by bacterial expression, demonstrated to be equipotent with PKIbeta-70, and also now demonstrated to occur in vivo. By Western blot analyses, six additional species of PKIbeta are also evident in tissues. Two of these represent the phospho forms of PKIbeta-78 and PKIbeta-70. The other four represent phospho and dephospho forms of two higher molecular mass PKIbeta species. These latter forms are currently termed PKIbeta-X and PKIbeta-Y, awaiting the full elucidation of their molecular identity. In adult rat testis and cerebellum, PKIbeta-70, PKIbeta-X, and PKIbeta-Y constitute 39, 23, and 32% and 15, 29, and 54% of the total tissue levels, respectively. In adult rat testis, 35-42% of each of these three species is present as a monophospho form, whereas no phosphorylation of them is evident in cerebellum. PKIbeta-78 is present at much lower levels in both rat testis and cerebellum (approximately 6 and 2% of the total, respectively) and almost entirely as a monophospho species. PKIbeta-78, like PKIbeta-70, is a high affinity and specific inhibitor of the cAMP-dependent protein kinase. PKIbeta-Y and PKIbeta-X, in contrast, also significantly inhibit the cGMP-dependent protein kinase.

Amino Acid Sequence↗

PubMed2000

Quantification analysis of translation initiation signal in vertebrate mRNAs: effect of nucleotides at positions +4(-)+6 upon efficiency of translation initiation.

Concerning the translation initiation signal in vertebrate mRNAs, a consensus, sequence, (GCC)GCC(A or G) CCATGG, has been proposed, but actual initiation sequences differ from it in a greater or lesser degree. Kozak monitored selection by ribosomes of the first versus second ATG codons as a function of mutations introduced at positions +4, and +6 of the first ATG codon. Codons possessing G at +4 strongly enhanced selection of the first ATG codon. However, ATG codon recognition was unaffected by most mutations in positions and +6. These data were well understood by our quantification analysis.

Animals↗

PubMed2004

Structure of the archaeal translation initiation factor aIF2 beta from Methanobacterium thermoautotrophicum: implications for translation initiation.

aIF2 beta is the archaeal homolog of eIF2 beta, a member of the eIF2 heterotrimeric complex, implicated in the delivery of Met-tRNA(i)(Met) to the 40S ribosomal subunit. We have determined the solution structure of the intact beta-subunit of aIF2 from Methanobacterium thermoautotrophicum. aIF2 beta is composed of an unfolded N terminus, a mixed alpha/beta core domain and a C-terminal zinc finger. NMR data shows the two folded domains display restricted mobility with respect to each other. Analysis of the aIF2 gamma structure docked to tRNA allowed the identification of a putative binding site for the beta-subunit in the ternary translation complex. Based on structural similarity and biochemical data, a role for the different secondary structure elements is suggested.

Amino Acid Sequence↗

PubMed1991

Gamma-subunits of G proteins, but not their alpha- or beta-subunits, are polyisoprenylated. Studies on post-translational modifications using in vitro translation with rabbit reticulocyte lysates.

Lipid modifications that may be introduced into several subunits of G proteins were explored by in vitro translation of recombinant mRNAs in reticulocyte lysates. In agreement with studies by others, myristic acid was incorporated into alpha i's and alpha o, but not alpha s, beta, or gamma's. In contrast, mevalonate (Mev) was incorporated only into gamma-subunits. Both, the gamma-subunit of transducin (gamma T) and that of other G proteins (gamma G) were modified by the lysates but with different characteristics. Labeled gamma T was unstable and was rapidly proteolyzed. Labeled gamma G was stable. The Mev-derivative in gamma G was sensitive to methyliodide and, after cleavage and chromatographic analysis, comigrated with the C20 polyisoprenol geranylgeraniol. This indicated that gamma G had been geranylgeranylated and that this polyisoprenoid was attached to the protein through a thioether linkage. It is thought that polyisoprenylation is defined by the COOH-terminal sequence Cys-A-A-X, where A is an aliphatic acid and X is any amino acid. Replacement by mutation of the Cys of the COOH-terminal -Cys-Ala-Ile-Leu sequence of gamma G with Ser abolished Mev incorporation, suggesting this Cys as the site of attachment of the geranylgeranyl moiety. Yet, Mev incorporation was less than 10% as much into gamma G with the Cys-A-A-X sequence -Cys-Ala-Ile-Trp. Consistent with geranylgeranylation, the C15 farnesyl moiety of farnesyl pyrophosphate was not incorporated into gamma G unless the incubations were fortified with Mev. In contrast, the farnesyl moiety was incorporated in an Mev-independent manner into gamma T (COOH terminus: -Cys-Val-Ile-Ser) and c-Ha-ras (COOH terminus: -Cys-Val-Leu-Ser) which are both farnesylated rather than geranylgeranylated. Thus, 1) separate enzymes appear to be involved in transferring farnesyl and geranylgeranyl groups to proteins, 2) structural factors other than the CAAX box contribute to the activity of the polyisoprenylating enzymes, and 3) this type of lipidation may be part of a proteolytic signaling system. Polyisoprenylation, which increases hydrophobicity of the derivatized protein, may play a role in anchoring not only ras but also G proteins to membranes.

Animals↗

PubMed1984

Regulation of IgM and IgD synthesis in B lymphocytes. II. Translational and post-translational events.

Studies investigating the relative rates of biosynthesis of mu- and delta-polypeptide chains in normal resting B lymphocytes have shown that the translation rate of mu m is about sevenfold higher than that of delta m, thus reflecting the relative abundance of mRNA encoding these two chains. The turnover rate of cell surface IgM is faster, however, than that of cell surface IgD, resulting in higher expression of cell surface IgD relative to IgM under steady state conditions. LPS stimulation of B lymphocytes induces the complete cessation of synthesis of the delta-chain, thus accounting for the gradual disappearance of IgD from the cell surface of activated cells.

Animals↗

PubMed1998

Evolutionary relationship between translation initiation factor eIF-2gamma and selenocysteine-specific elongation factor SELB: change of function in translation factors.

Eubacterial and eukaryotic translation initiation systems have very little in common, and therefore the evolutionary events that gave rise to these two disparate systems are difficult to ascertain. One common feature is the presence of initiation, elongation, and release factors belonging to a large GTPase superfamily. One of these initiation factors, the gamma subunit of initiation factor 2 (eIF-2gamma), is found only in eukaryotes and archaebacteria. We have sequenced eIF-2gamma gene fragments from representative diplomonads, parabasalia, and microsporidia and used these new sequences together with new archaebacterial homologues to examine the phylogenetic position of eIF-2gamma within the GTPase superfamily. The archaebacterial and eukaryotic eIF-2gamma proteins are found to be very closely related, and are in turn related to SELB, the selenocysteine-specific elongation factor from eubacteria. The overall topology of the GTPase tree further suggests that the eIF-2gamma/SELB group may represent an ancient subfamily of GTPases that diverged prior to the last common ancestor of extant life.

Amino Acid Sequence↗

PubMed1983

Localization of the target site for translational regulation of the L11 operon and direct evidence for translational coupling in Escherichia coli.

The L11 ribosomal protein operon in Escherichia coli consists of the structural genes for proteins L11 and L1. Hybrid deletion plasmids were constructed carrying these two genes with decreasing amounts of the leader mRNA under lac transcriptional control. Measurements of mRNA and protein synthesis directed by these plasmids in vitro and in vivo demonstrate that the regulation of this operon is posttranscriptional and identifies a region of the mRNA, preceding the proximal L11 gene, important for successful feedback inhibition of L11 and L1 synthesis by L1. Additionally, deletions extending to the ribosome binding site of the L11 gene fail to synthesize both L11 and the downstream L1 protein although synthesis of the corresponding mRNA remains unchanged. These results directly demonstrate the presence of translational coupling in this bicistronic operon.

Bacterial Proteins↗

PubMed1992

Regulation of translation and proteolysis during the development of embryonic dorso-ventral polarity in Drosophila. Homology of easter proteinase with Limulus proclotting enzyme and translational activation of Toll receptor synthesis.

The generation of dorso-ventral polarity during Drosophila embryogenesis is regulated by the action of 12 maternally expressed gene products, the dorsal group. These products act together to form a dorso-ventral nuclear gradient of the transcription factor dorsal. At least three of the dorsal group genes (snake, easter and gastrulation defective) encode secreted serine proteinases which probably function during early development in the perivitelline compartment of the embryo. Here, we report that the easter proteinase is homologous in its light chain sequence to the haemocyte proclotting enzyme (PCE) of the Japanese horseshoe crab Tachypleus tridentatus. PCE is the terminal member of a proteolytic cascade activated in response to microbial polysaccharides and acts to cleave coagulogen, an invertebrate equivalent of fibrinogen. On the basis of this homology we are able to predict with confidence the overall primary structure of the easter proteinase, its mode of activation and its substrate specificity. The result also suggests that easter functions zygotically in haemocytes in a Drosophila defence response analogous to that found in Tachypleus. We also show here that the Toll receptor protein is absent in early cleavage embryos but accumulates rapidly at the syncitial blastoderm stage, the developmental stage at which its function is required. This finding suggests that translation of Toll mRNA is regulated in response to fertilisation and egg deposition. These two observations are consistent with a model of dorso-ventral pattern formation in which a proteolytic cascade is activated uniformly in the perivitelline compartment of the embryo and causes the release of ventrally localised ligands of the Toll receptor. A possible alternative model in which a proteolytic cascade is activated in response to a ventrally restricted signal is also discussed.

Amino Acid Sequence↗

PubMed2002

Expression of the Escherichia coli pcnB gene is translationally limited using an inefficient start codon: a second chromosomal example of translation initiated at AUU.

Expression of the gene pcnB, encoding the dispensable Escherichia coli poly(A) polymerase (PAPI), which is toxic when overproduced, was investigated. Its promoter was identified and found to be moderately strong when used to express a beta-galactosidase reporter. Expression levels were not affected by increasing or decreasing PcnB concentration. Translation of pcnB was found to initiate from the non-canonical initiation codon AUU. The only other coli gene reported to use AUU as initiation codon is infC, which encodes the initiation factor IF-3. AUU, in common with other rarely used initiation codons, is discriminated against by IF-3, resulting in the aborting of most AUU-promoted initiation events. This enables AUU to form part of an autoregulatory circuit controlling IF-3 production. We show that InfC discrimination reduces PcnB production fivefold. This is the first instance of this mechanism being used to limit severely the production of a potentially toxic product.

Base Sequence↗

PubMed1996

Evidence for developmentally regulated transcriptional, translational and post-translational control of metallothionein gene expression in hair follicles.

The distribution of metallothionein (MT) and MT mRNAs was examined in hair (wool) follicles, where high levels of cell proliferation are found and where the resulting cells provide a temporal record of differentiation events. MT was found in the cytoplasm and some nuclei of follicle bulb cells of the proliferative zone, outer root sheath cells and in basal layer cells of sebaceous glands and sweat glands. The population of 5-bromo-2'-deoxyuridine (BrdU)+ cells in these tissues overlapped, but were not completely coincident with the distribution of MT staining. MT mRNA expression in hair (wool) follicles was assessed by in situ hybridization with four gene-specific sheep MT (sMT) isoforms. Intense signals were obtained with the sMT-Ib probe in follicle bulb cells from the proliferative zone to the keratogenous zone. Signals from the sMT-Ia probe were present in the same cells, but were much weaker. No signals were detected using the sMT-Ic and sMT-II gene-specific probes. The findings suggest that: (1) MT is important in cell proliferation and/or cell differentiation in the hair follicle bulb; (2) MT translation is inhibited during cell differentiation and migration; and (3) tissue-specific expression of uncharacterized sMT isoforms is likely.

Animals↗

PubMed1974

Separation of specific initiation factors involved in the translation of myosin and myoglobin messenger RNAs and the isolation of a new RNA involved in translation.

Two messenger specific factors, phosphocellulose fractions 3 and 4, have been isolated from the initiation factor 3 fraction of red muscle initiation factors by chromatography on phosphocellulose. When added to a reticulocyte cell-free system containing both myoglobin and myosin mRNAs, phosphocellulose fraction 3 is found to specifically stimulate the synthesis of myoglobin while phosphocellulose fraction 4 is found to specifically stimulate the synthesis of myosin. In addition, a new RNA, isolated from the initiation factor 3 fraction, is shown to specifically inhibit the translation of heterologous mRNAs.

Animals↗