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PubMed1998

Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase.

A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate aminotransferase (AAT; EC 2.6.1.1) was isolated. Genomic Southern blots and analysis of genomic clones indicated pSAT1 was encoded by a single copy gene. pSAT1 contained an open reading frame with ca. 90% amino acid identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2. Alignment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with characteristics of a peroxisomal targeting signal, designated PTS2, including the modified consensus sequence RL-X5-HF. The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT cDNAs. Plasmids constructed to express the open reading frame initiated by each of the putative start codons produced proteins with AAT activity in Escherichia coli. Immune serum raised against the pSAT1-encoded protein reacted with three soybean AAT isozymes, AAT1 (glyoxysomal), AAT2 (cytosolic), and AAT3 (subcellular location unknown). We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and the cytosolic isozyme AAT2 is produced by translational initiation from ATG2. N-terminal sequencing of purified AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the PTS2. Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to generate both cytosolic and glyoxysomal or peroxisomal forms of AAT.

Amino Acid Sequence↗

PubMed2002

Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments.

Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a 'classical' Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9 kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99 bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product.

Amino Acid Sequence↗

PubMed1997

Bovine coronavirus I protein synthesis follows ribosomal scanning on the bicistronic N mRNA.

The mRNA encoding the 49-kDa nucleocapsid protein (N) of the bovine coronavirus is bicistronic. A 23-kDa protein, termed the I protein for the 'internal' open reading frame (ORF), is also synthetized but in the +1 reading frame beginning 61 nt downstream of the N start codon. Sequences flanking the N and I start codons suggest that the I ORF might be accessed by scanning ribosomes passing over the N start codon. Here we test this idea and demonstrate with translation studies both in vitro and in vivo that the I protein is synthesized according to the leaky scanning model for initiation of translation on the subgenomic N mRNA molecule.

Animals↗

PubMed1999

Computer analyses of complete genomes suggest that some archaebacteria employ both eukaryotic and eubacterial mechanisms in translation initiation.

The translation initiation mechanism of archaebacteria is still not clearly understood. Our previous work showed that ATG triplets before start codons have been strongly depleted in eukaryotic genomes, presumably because ribosome of eukaryotes scans mRNA from the 5' to 3' direction to find proper start codons. Extra ATG triplets before start codons would confuse the process and thus they have been negatively selected in eukaryotic genomes. In eubacterial genomes, on the other hand, ribosome binds to the Shine-Dalgarno (SD) sequence at once without mRNA scanning, and the characteristic patterns of ATG triplet depletion were not observed (Saito, R., Tomita, M., 1999. On negative selection against ATG triplets near start codons in eukaryotic and procaryotic genomes. J. Mol. Evol. 48, 213-217). The ATG triplet analysis on archaebacterial genomes revealed that Methanococcus jannaschii and Pyrococcus horikoshii show patterns similar to eukaryotes, implying that these species employ scanning of mRNA from the 5' to 3' direction in the process of translation initiation. On the other hand, our earlier study found that these archaea have SD-like sequences, which are complementary to the 3' end sequence of 16S rRNA, as in eubacterial translation initiation (Osada, Y., Saito, R., Tomita, M. Analysis of base-pairing potentials between 16S rRNA and 5' UTR for translation initiation in various procaryotes. Bioinformatics, in press). These two results combined lead us to conclude that these archaea probably use a hybrid mechanism; their ribosome scans mRNAs from the 5' to 3' direction and then 16S rRNA binds to the SD-like sequence of the 5' UTR.

5' Untranslated Regions↗

PubMed1990

Initiation of translation at CUG, GUG, and ACG codons in mammalian cells.

Site-directed mutants of the ACG start codon of the C' protein encoded in the polycistronic Sendai virus P/C mRNA revealed that CUG, GUG, and ACG codons initiated translation rather efficiently (10-30% of the AUG initiation) in COS-1 host cells. In addition, AUA and AUU codons initiated translation at about 5% efficiency, while UUG did not initiate translation. The sequence context of these start codons (purine residues at -3 and +4) was crucial in their recognition by the ribosome. The location of the non-AUG codons in the P/C mRNA did not play a role in its recognition by ribosomes. By using CUG, the most efficient non-AUG start codon, instead of the original ACG codon and inserting an additional upstream CUG codon in the P/C mRNA, the amount of the C' protein was increased and a novel protein was synthesized. Syntheses of an increased level of C' and the novel protein did not affect downstream initiations of the P and C proteins, suggesting that more ribosomes bind the mRNA than are actually utilized for initiation of translation.

Animals↗

PubMed1999

The short 5' untranslated region of the betaA3/A1-crystallin mRNA is responsible for leaky ribosomal scanning.

Leaky ribosomal scanning allows the expression of multiple proteins from a single mRNA by occasionally skipping the first start codon, and initiating translation at a subsequent one. BetaA3- and betaA1-crystallin, two members of the beta-crystallin family of vertebrate eye lens proteins, are produced via this mechanism, of which, until now, only very few examples have been found in eukaryotic genes. Since the two start codons on the betaA3/A1 messenger lie in the same reading frame, the two translated proteins are identical, except for the 17 residues shorter N-terminal extension of betaA1-crystallin. It has been suggested that the very short leader (5-7 nucleotides) of the betaA3/A1 messenger might cause slippage at the first start codon, although the unfavorable context of this start codon might also be responsible. Using transient transfections, we now demonstrate that increasing the length of the leader sequence to 67 nucleotides indeed completely abolishes translation initiation at the second start codon, and thus expression of the betaA1-crystallin protein. Messengers having a leader of 5, 7 or 14 nucleotides all express both betaA3- and betaA1-crystallin at very similar relative levels.

5' Untranslated Regions↗

PubMed1990

The 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii. 2. Molecular cloning and sequence analysis of the gene encoding the succinyltransferase component.

The nucleotide sequence encoding the succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. Previously the cloning in Escherichia coli of the gene encoding lipoamide dehydrogenase from A. vinelandii was reported [Westphal, A.H. & de Kok, A. (1988) Eur. J. Biochem. 172, 299-305]. The 3.2-kb fragment used for the sequence determination contained the main part of the gene encoding succinyltransferase. The complete E2o gene, as well as the gene encoding the 2-oxoglutarate dehydrogenase component, resided on a 14.7-kb fragment from which the 3.2-kb fragment was subcloned. The protein-coding sequence of the gene consists of 1200 bp (400 codons, including the AUG start codon and the UGA stop codon). It is separated from the gene encoding the 2-oxoglutarate dehydrogenase component by 42 bp. No E. coli-like promoter sequence was found. A putative ribosome-binding site is located 9-15 bp upstream from the start codon. No terminator sequences were found downstream of the stop codon. This makes it likely that the three genes of the oxoglutarate dehydrogenase complex are transcribed as a single mRNA transcript analogous to the pyruvate dehydrogenase complex in E. coli. The intact gene was subcloned from the 14.7-kb fragment and brought to high expression under the influence of the vector-encoded lacZ promoter. The similarity with the E. coli enzyme is high with 63% identity. Like the enzyme from E. coli, it consists of a single lipoyl-binding domain, a putative E1- and E3-binding domain and a catalytic domain. The main difference is found in a 31-residue sequence rich in alanine and proline located between the lipoyl domain and the putative E1- and E3-binding domain. This sequence, usually found in acetyltransferases and there identified as a highly mobile region by 1H-NMR, is replaced by a more polar, charged region in the E. coli enzyme.

Acyltransferases↗

PubMed1991

Distance-dependent translational coupling and interference in Lactococcus lactis.

The possibility of raising the expression level of a heterologous gene in Lactococcus lactis by exploiting the principle of translational coupling was investigated. For this purpose, the Escherichia coli lacZ gene was transcriptionally fused to a short open reading frame (ORF) of lactococcal origin. A Shine-Dalgarno (SD) sequence was introduced at the boundary of the two ORFs. In a series of otherwise identical plasmids, the relative positions of the translational stop codon of the upstream ORF and the translational start codon of the downstream ORF (lacZ) were varied. The expression of lacZ gradually increased as the stop and start codons were placed in closer proximity. A concomitant switch from translational interference to translational coupling was observed. Best results were obtained with partially overlapping stop and start codons. It is concluded that the principle of translational coupling offers good possibilities to increase the level of heterologous gene expression in L. lactis.

Amino Acid Sequence↗

PubMed1990

The 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii. 1. Molecular cloning and sequence analysis of the gene encoding the 2-oxoglutarate dehydrogenase component.

The nucleotide sequence of the gene encoding the 2-oxoglutarate dehydrogenase component (E1o) of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. The protein-coding sequence consists of 2832 bp (944 codons, including the AUG start codon and the UAA stop codon). The predicted molecular mass (105,687 Da) is in good agreement with that published for the isolated enzyme. The E1o gene is separated from the gene encoding the E2o component by a 42-bp intergenic region. No Escherichia-coli-like promoter sequences are found in the sequenced 97 bp upstream from the E1o gene. A putative ribosome-binding site is located 10-16 bp upstream from the start codon of the E1o gene. No terminator sequences could be detected downstream from the stop codon. Together with the identical situation for the E2o gene and the presence of terminating sequences downstream of the E3 gene, it can be assumed that all three genes of the 2-oxoglutarate dehydrogenase multienzyme complex are transcribed as a single mRNA transcript under the control of a promoter, located more than 100 bp upstream of the E1o gene, analogous to the pyruvate dehydrogenase complex in E. coli. The similarity with the sucA gene of E. coli is high with 59% identity.

Amino Acid Sequence↗

PubMed1988

Molecular clones of alpha-tubulin genes of Plasmodium yoelii reveal an unusual feature of the carboxy terminus.

By using a Trypanosoma brucei alpha-tubulin cDNA probe under reduced stringency hybridization conditions, we have isolated two genomic clones that constitute portions of alpha-tubulin genes of the rodent malarial parasite Plasmodium yoelii. P. yoelii has two alpha-tubulin genes, the 3' portions of which were present in the two clones, Py alpha T1 and Py alpha T2, containing 1.3 kb and 6.6 kb EcoRI fragments respectively. The 1358 bp Py alpha T1 clone was completely sequenced and found to contain 591 nucleotides of uninterrupted coding sequence with a strong bias for AT-rich codons, starting with codon 254 of a consensus alpha-tubulin sequence. Numerous attempts to clone 5' portions of these genes were unsuccessful. A single mRNA of 2.3 kb was recognized by both the clones in the erythrocytic stages of P. yoelii. A probe constituting the untranslated sequences of Py alpha T1 also recognized this RNA but failed to hybridize with Py alpha T2 sequences, indicating that the gene represented by the Py alpha T1 clone was expressed during the erythrocytic stages. The deduced amino acid sequence of the Py alpha T1 gene terminates in Tyr-Glu instead of Glu-Tyr observed in alpha-tubulins of almost all other organisms. The difference observed may have implications for alpha-tubulin metabolism in malarial parasites.

Amino Acid Sequence↗

PubMed1986

Cloning, expression and complete nucleotide sequence of the Bacillus stearothermophilus L-lactate dehydrogenase gene.

The structural gene for L-lactate dehydrogenase (LDH; EC 1.1.1.27) from Bacillus stearothermophilus NCA 1503 has been cloned in Escherichia coli and its complete nucleotide sequence determined. The predicted amino acid (aa) sequence of the LDH enzyme agrees with the previously determined aa sequence except to three positions: aa 125 and 126, Ser-Glu, are inverted whilst His at position 130 has been replaced by Ser in our sequence. The lct gene consists of an open reading frame (ORF) commencing from the ATG start codon of 951 bp followed by a TGA stop codon. Upstream from the start codon is a strong (delta G = -14.4 kcal) Shine-Dalgarno (SD) sequence, a feature typical of Gram-positive ribosome binding sites. Putative RNA polymerase recognition signals (-35 and -10 regions) have been identified upstream from the lct structural gene but there are no structures resembling Rho-independent transcription termination signals downstream from the TGA stop codon. Two further ORFs, preceded by SD sequences, are present downstream from the lct gene. Thus the lct gene may constitute the first gene of an operon. Subclones of the lct gene have been constructed in the expression plasmid pKK223-3 and the LDH enzyme produced in soluble form at levels of up to 36% of the E. coli soluble cell protein.

Amino Acid Sequence↗

PubMed1994

The dipeptide permease of Escherichia coli closely resembles other bacterial transport systems and shows growth-phase-dependent expression.

The dipeptide permease (Dpp) of Escherichia coli transports peptides consisting of two or three L-amino acids. The periplasmic dipeptide-binding protein (DBP), encoded by the dppA gene, also serves as a chemoreceptor. We sequenced the dpp locus, which comprises an operon of five genes, dppABCDE. Its organization is the same as the oligopeptide permease (opp) operon of Salmonella typhimurium and the spo0K operon of Bacillus subtilis. The dpp genes are also closely related to the hbpA gene, which encodes a haem-binding lipoprotein, and four other genes in an unlinked operon of unknown function in Haemophilus influenzae. Each Dpp protein has an Opp, Spo0K and H. influenzae homologue. Transcription of the dpp operon initiates 165 bases upstream of the predicted dppA start codon. The start site for transcription is preceded by potential -35 and -10 regions of a sigma 70 promoter. During exponential growth in Luria-Bertani (LB) broth, the level of dpp mRNA increases in two steps, one between A590 0.2 and 0.4 and one between A590 0.7 and 1.0. The 310 nucleotides between dppA and dppB include a RIP (repetitive IHF-binding palindromic) element, whose deletion from a multi-copy plasmid causes fivefold and 10-fold reductions in the levels of upstream and downstream dpp mRNA, respectively.

Amino Acid Sequence↗

PubMed1992

cis-acting elements involved in the alternative translation initiation process of human basic fibroblast growth factor mRNA.

Four forms of basic fibroblast growth factor (bFGF) are synthesized from the same mRNA, resulting from alternative initiations of translation at three CUG start codons and one AUG start codon. The CUG- and AUG-initiated forms have distinct intracellular localizations and can modify cell phenotypes differently, indicating that control of the alternative expression of the different forms of bFGF has an important impact on the cell. In this study, we investigated the roles of the mRNA 5' untranslated region and the alternatively translated region located between the CUG and AUG codons in the regulation of alternative translation of the different forms of bFGF. Deletions and site-directed mutagenesis were carried out in bFGF mRNA leader, and translation was studied in vitro and in vivo. The results enabled us to identify five cis-acting RNA elements (two in the 5' untranslated region and three in the alternatively translated region) involved in the regulation of either global or alternative initiation of translation. Each of these elements had a specific effect on the level of synthesis of the different forms of bFGF. Furthermore, we showed that the 5' untranslated region regulatory elements had different effects on bFGF translation, depending on the translation system used. These results suggest that bFGF translation is modulated by cis-acting elements corresponding to secondary or tertiary RNA structures, which could be the targets of cell-specific trans-acting factors.

Animals↗

PubMed1995

Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes.

Alternative initiations of translation of the human fibroblast growth factor 2 (FGF-2) mRNA, at three CUG start codons and one AUG start codon, result in the synthesis of four isoforms of FGF-2. This process has important consequences on the fate of FGF-2: the CUG-initiated products are nuclear and their constitutive expression is able to induce cell immortalization, whereas the AUG-initiated product, mostly cytoplasmic, can generate cell transformation. Thus, the different isoforms probably have distinct targets in the cell. We show here that translation initiation of the FGF-2 mRNA breaks the rule of the cap-dependent ribosome scanning mechanism. First, translation of the FGF-2 mRNA was shown to be cap independent in vitro. This cap-independent translation required a sequence located between nucleotides (nt) 192 and 256 from the 5' end of the 318-nt-long 5' untranslated region. Second, expression of bicistronic vectors in COS-7 cells indicated that the FGF-2 mRNA is translated through a process of internal ribosome entry mediated by the mRNA leader sequence. By introducing additional AUG codons into the RNA leader sequence, we localized an internal ribosome entry site to between nt 154 and 318 of the 5' untranslated region, just upstream of the first CUG. The presence of an internal ribosome entry site in the FGF-2 mRNA suggests that the process of internal translation initiation, by controlling the expression of a growth factor, could have a crucial role in the control of cell proliferation and differentiation.

Animals↗

PubMed1987

[Duplication of a synthetic gene for human leukocyte interferon and its expression in polycistron mRNA with coupled translation system].

Using a chemically synthesised adapter, the coding part of an artificial gene for human leukocyte alpha 2 interferon (ifn-alpha 2) has been duplicated. The adapter contained a termination signal of the first gene (TAA) within the Shine-Dalgarno sequence of the second gene (TAAGGA), distance between the terminating codon and starting codon of the second gene being 11 nucleotides. In another case this distance was 69 nucleotides, with the same SD sequence. The expression of the tandems as a part of polycistrons has been studied under control of promoters Plac, (Ptrp)2 of E. coli, and PVIII of M13 phage. It was found that tandems of ifn-alpha 2 genes in polycistronic structures trp L-ifn-ifn and IX-VIII-ifn-ifn under control of promoters (Ptrp)2 and PVIII, respectively, provided high level of the interferon biosynthesis, thus differing from the tandem under Plac promoter control, which had only ifn-ifn translation coupling.

Escherichia coli↗

PubMed1989

Restoration of a translational stop-start overlap reinstates translational coupling in a mutant trpB'-trpA gene pair of the Escherichia coli tryptophan operon.

The trpB and trpA coding regions of the polycistronic trp mRNA of Escherichia coli are separated by overlapping translation stop and start codons. Efficient translation of the trpA coding region is subject to translational coupling, i.e., maximal trpA expression is dependent on prior translation of the trpB coding region. Previous studies demonstrated that the trpA Shine-Dalgarno sequence (within trpB) and/or the location of the trpB stop codon influenced trpA expression. To examine the effect of stop codon location specifically, we constructed plasmids in which different nucleotide sequences preceding the trpA start codon were retained, and only the reading frame was changed. When trpB translation proceeded in the wild type reading frame and terminated at the normal trpB stop codon, trpA polypeptide levels were elevated over the levels observed when translation stopped before or after the natural trpB stop codon. The proximity of the trpB stop codon to the trpA start codon therefore markedly influences trpA expression.

Base Sequence↗

PubMed1992

Both forms of translational initiation factor IF2 (alpha and beta) are required for maximal growth of Escherichia coli. Evidence for two translational initiation codons for IF2 beta.

The gene infB codes for two forms of translational initiation factor IF2; IF2 alpha (97,300 Da) and IF2 beta (79,700 Da). IF2 beta arises from an independent translational event on a GUG codon located 471 bases downstream from IF2 alpha start codon. By site-directed mutagenesis we constructed six different mutations of this GUG codon. In all cases, IF2 beta synthesis was variably affected by the mutations but not abolished. We show that the residual expression of IF2 beta results from translational initiation on an AUG codon located 21 bases downstream from the mutated GUG. Furthermore, two forms of IF2 beta have been separated by fast protein liquid chromatography and the determination of their N-terminal sequences indicated that they resulted from two internal initiation events, one occurring on the previously identified GUG start codon, the other on the AUG codon immediately downstream. We conclude that two forms of IF2 beta exist in the cell, which differ by seven aminoacid residues at their N terminus. Only by mutating both IF2 beta start codons could we construct plasmids that express only IF2 alpha. A plasmid expressing only IF2 beta was obtained by deletion of the proximal region of the infB gene. Using a strain that carries a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic lambda phage, we could cure the lambda phage when the plasmids expressing only one form of IF2 were supplied in trans. We found that each one of the two forms of IF2, at near physiological levels, can support growth of Escherichia coli, but that growth is retarded at 37 degrees C. This result shows that both forms of IF2 are required for maximal growth of the cell and suggests that they have acquired some specialized but not essential function.

Amino Acid Sequence↗

PubMed1989

Overlap between pdxA and ksgA in the complex pdxA-ksgA-apaG-apaH operon of Escherichia coli K-12.

We report that pdxA, which is required for de novo biosynthesis of pyridoxine (vitamin B6) and pyridoxal phosphate, belongs to an unusual, multifunctional operon. The pdxA gene was cloned in the same 3.5-kilobase BamHI-EcoRI restriction fragment that contains ksgA, which encodes the 16S rRNA modification enzyme m6(2)A methyltransferase, and apaH, which encodes diadenosine tetraphosphatase (ApppA hydrolase). Previously, Blanchin-Roland et al. showed that ksgA and apaH form a complex operon (Mol. Gen. Genet. 205:515-522, 1986). The pdxA gene was located on recombinant plasmids by subcloning, complementation, and insertion mutagenesis, and chromosomal insertions at five positions upstream from ksgA inactivated pdxA function. DNA sequence analysis and minicell translation experiments demonstrated that pdxA encoded a 35.1-kilodalton polypeptide and that the stop codon of pdxA overlapped the start codon of ksgA by 2 nucleotides. The translational start codon of pdxA was tentatively assigned based on polypeptide size and on the presence of a unique sequence that was also found near the translational start of PdxB. This conserved sequence may play a role in translational control of certain pyridoxine biosynthetic genes. RNase T2 mapping of chromosomal transcripts confirmed that pdxA and ksgA were members of the same complex operon, yet about half of ksgA transcripts arose in vivo under some culture conditions from an internal promoter mapped near the end of pdxA. Transcript analysis further suggested that pdxA is not the first gene in the operon. These structural features support the idea that pyridoxine-biosynthetic genes are members of complex operons, perhaps to interweave coenzyme biosynthesis genetically with other metabolic processes. The results are also considered in terms of ksgA expression.

Amino Acid Sequence↗