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PubMedDate not supplied

Initiation of translation at internal AUG codons in mammalian cells.

Initiation of translation of eukaryotic mRNAs typically occurs at the first AUG triplet from the 5' end of the message, although several notable exceptions have been described. Using vectors which efficiently express the gene encoding the surface antigen of hepatitis B virus in monkey cells, we have studied the consequences of inserting ATG triplets in all three reading frames upstream of the usual translational initiation codon of this gene. In agreement with the scanning model for eukaryotic translation initiation, these additional codons can severely depress the initiation of translation at the 'authentic' start codon, although the extent of inhibition depends on sequences flanking the upstream AUG. Such inhibition can, however, be at least partially suppressed by the presence of a translation termination codon in-frame with the upstream AUG. These results raise the possibility that mammalian ribosomes can reinitiate translation at an AUG codon after previously initiating, and terminating, at an upstream site.

Animals↗

PubMed1998

Synthesis in Escherichia coli of two smaller enzymically active analogues of Coxiella burnetii macrophage infectivity potentiator (CbMip) protein utilizing a single open reading frame from the cbmip gene.

FK506-binding proteins (FKBPs) have been identified in a variety of eukaryotic and prokaryotic organisms. Macrophage infectivity potentiator (CbMip, 23.5 kDa) protein of the obligate intracellular bacterium, Coxiella burnetii, was shown previously to belong to the family of FKBPs based on sequence homology and peptidyl-prolyl cis/trans isomerase (PPIase) activity. Further characterization of the cbmip gene has identified two additional proteins with molecular masses of 15.5 and 15.0 kDa that are synthesized, in addition to the 23.5 kDa CbMip, when expressed in Escherichia coli. Amino acid sequencing at the N-terminus combined with transcription and translation fusion expression revealed that the two proteins were synthesized from the same open reading frame of the cbmip gene, but starting at different internal translation start codons, probably by translational reinitiation. When the internal methionines serving as start sites were replaced with lysine by site-directed mutagenesis, the synthesis of 15.5 and 15.0 kDa proteins was abolished even though the synthesis of 23.5 kDa CbMip was intact. This confirmed that the 15.5 and 15.0 kDa proteins are indeed generated by translational reinitiation and are not degradation products of the 23.5 kDa protein. Like other FKBPs, both 15.5 and 15.0 kDa proteins exhibit PPIase activity. Because they share significant sequence homology with FKBPs and have a similar PPIase activity, 15.5 and 15. 0 kDa proteins are designated as C. burnetii FKBP (Cb-FKBP) analogues I and II, respectively. TnphoA mutagenesis demonstrated that whereas the large protein (CbMip) is secreted, Cb-FKBP analogues I and II are cytoplasmic, indicating that structural variations could allow for different subcellular compartmentalization of similar proteins. Western-blot analysis of lysates of purified C. burnetii using a CbMip-specific monoclonal antibody revealed the presence of a protein migrating at approximately 15 kDa, indicating the presence of smaller Cb-FKBP analogue(s) in C. burnetii, although at much lower levels compared with 23.5 kDa CbMip. This unique gene organization seen with cbmip may provide the organism with a mechanism of efficient use of its limited genetic information to synthesize proteins that are structurally different yet functionally similar.

Amino Acid Sequence↗

PubMed2000

Optimizing heterologous expression in dictyostelium: importance of 5' codon adaptation.

Expression of heterologous proteins in Dictyostelium discoideum presents unique research opportunities, such as the functional analysis of complex human glycoproteins after random mutagenesis. In one study, human chorionic gonadotropin (hCG) and human follicle stimulating hormone were expressed in Dictyostelium. During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start codon. The Dictyostelium genome has a higher AT content than the human, resulting in a different codon preference. The hCG-beta gene contains three clusters with infrequently used codons that were changed to codons that are preferred by Dictyostelium. The results reported here show that optimizing the first 5-17 codons of the hCG gene contributes to 4- to 5-fold increased expression levels, but that further optimization has no significant effect. These observations suggest that optimal codon usage contributes to ribosome stabilization, but does not play an important role during the elongation phase of translation. Furthermore, adapting the 5'-sequence of the hCG gene to the Dictyostelium 'Kozak'-like sequence increased expression levels approximately 1.5-fold. Thus, using both codon optimization and 'Kozak' adaptation, a 6- to 8-fold increase in expression levels could be obtained for hCG.

Amino Acid Sequence↗

PubMed2005

A single complementary-sense transcript of a geminiviral DNA beta satellite is determinant of pathogenicity.

Small circular single-stranded DNA satellites, termed DNAbeta, have recently been found associated with some geminivirus infections. The DNA beta associated with Cotton leaf curl virus is responsible for symptom expression of a devastating disease in Pakistan. Mutagenesis of DNA beta revealed that the complementary-sense open reading frame (ORF) betaC1 is required for inducing disease symptoms in Nicotiana tabacum. An ORF present on the virion-sense strand betaV1 appeared to have no role in pathogenesis. Tobacco plants transformed with a betaC1 ORF under the control of the Cauliflower mosaic virus 35S promoter or with a dimeric DNA beta exhibited severe disease-like phenotypes, while plants transformed with a mutated version of betaC1 appeared normal. Northern blot analysis of RNA from the transgenic plants, using strand-specific probes, identified a single complementary-sense transcript. The transcript carries the full betaC1 ORF encoding a 118-amino acid product. It maps to the DNA beta at nucleotide position 186 to 563 and contains a polyadenylation signal 18 nt upstream of the stop codon. A TATA box is located 43 nt upstream of the start codon. Our results indicate that betaC1 protein is responsible for DNA beta-induced disease symptoms.

Amino Acid Sequence↗

PubMed1999

Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria.

Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kanamycin resistance) or aacCI (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptII-TtrpA) and CAS-Gm (Ptac/PaacCI-aacCI-TtrpA) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-Ptac-nptII-TtrpA) or CAS-GGm (gusA-Ptac/PaacCI-aacCI-TtrpA) cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-nptII-TtrpA) and mTn5-Gm (containing Ptac/PaacCI-aacCI-TtrpA) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-Ptac-nptII-TtrpA) and mTn5-GGm (containing gusA-Ptac/PaacCI-aacCI-TtrpA) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid- or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other gram-negative bacteria.

Base Sequence↗

PubMed1995

Sequence analysis of the Streptococcus mutans Ingbritt dexA gene encoding extracellular dextranase.

The complete nucleotide sequence (3,747 bp) of the dextranase gene (dexA) and flanking regions of the chromosome of Streptococcus mutans Ingbritt (serotype c) were determined. The open reading frame for dexA was 2,550 bp, ending with a stop codon TGA. A putative ribosome-binding site, promoter preceding the start codon, and potential stem-loop structure were identified. The presumed dextranase protein (DexA) consisting of 850 amino acids was estimated to have a molecular size of 94,536 Da and a pI of 4.79. The nucleotide sequence and the deduced amino acid sequences of S. mutans dexA exhibited homologies of 57.8% and 47.0%, respectively, to those of Streptococcus sobrinus dex. The homologous region of dex of S. sobrinus was in the N-terminal half. The C terminus of DexA consisted of a hexapeptide LPQTGD, followed by 7 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged hexapeptide tail, which have been reported as a common structure of C termini of not only the surface-associated proteins of Gram-positive cocci but also the extracellular enzymes such as beta-fructosidase of S. mutans and dextranase of S. sobrinus. The DexA protein had no significant homology with the glucosyltransferases, the glucan-binding protein, or the dextranase inhibitor of mutans streptococci.

Amino Acid Sequence↗

PubMed2006

Posttranscriptional control of the Salmonella enterica flagellar hook protein FlgE.

Previous work suggested that the FlgE (flagellar hook subunit) protein in Salmonella enterica serovar Typhimurium was posttranscriptionally regulated in response to the stage of flagellar assembly. Specifically, the FlgE protein could be detected in flagellar mutants defective at the stages of assembly before or after rod assembly but not in rod assembly mutants, yet flgE mRNA levels were unaffected. To elucidate posttranscriptional mechanisms involved in the coupling of flgE gene expression to hook assembly, the RNA sequences at the 5' and 3' ends of the flgE-containing mRNA processed from the large flgBCDEFGHIJKL operon were determined by rapid amplification of cDNA ends, and secretion of the FlgE protein in different flagellar assembly mutant strains was analyzed. The sequences 5' and 3' of the flgE gene where RNA processing occurred was within 15 bases upstream of the flgD stop codon and at bases 145 to 147 downstream of the flgF start codon, respectively. The ribosome binding site of the flgD gene was found to be inhibitory to flgE translation in strains deleted for the upstream flgD gene, unless the region 15 bases upstream of the flgD stop codon was present. Secretion of FlgE into the periplasm was monitored using beta-lactamase (Bla) fusions as a periplasm-specific reporter, which conferred resistance to ampicillin when FlgE-Bla was secreted into the periplasm. Using this assay, we found that the effect of rod assembly mutants on FlgE levels was due to FlgE turnover in the periplasm and that the FliE rod component protein was required for efficient FlgE-Bla secretion.

Bacterial Proteins↗

PubMed1981

Factors governing the expression of a bacterial gene in mammalian cells.

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.

Animals↗

PubMed1988

Structure and evolution of bacterial adenylate cyclase: comparison between Escherichia coli and Erwinia chrysanthemi.

The cya genes, coding for adenylate cyclase, from Escherichia coli and Erwinia chrysanthemi B374 are compared after determination of a 3632 bp long nucleotide sequence of the hemC-cya region of E. chrysanthemi, encompassing the whole cya gene. In spite of a large divergence between the two organisms, especially visible in non coding regions, the amino acid sequence of the proteins are very similar, except at the very distal carboxyl end. Codon usage is different in the two organisms, and E. chrysanthemi tends to restrict translation to codons ending in G or C. Conservation of the translation initiation start region (including the poor ribosome binding site GGCG, and the TTG start codon), suggests that a specific protein synthesis process controls adenylate cyclase expression. Finally a palindromic unit, of primary sequence differing from the E. coli counterpart, borders the gene in E. chrysanthemi.

Adenylyl Cyclases↗

PubMed2001

Posttranscriptional regulation of human ADH5/FDH and Myf6 gene expression by upstream AUG codons.

Upstream open-reading frames are unusual in mammalian mRNAs. The 5' untranslated region of ADH5 mRNA contains an upstream open-reading frame (uORF) with two possible AUG start codons. Myf6 mRNA contains three tandem AUG repeats at the translation start site, a rare feature. Mutation at one or both of the upstream AUG codons in the ADH5 mRNA increased gene expression twofold in CV-1, NIH/3T3, HeLa, and SL2 cells. Mutation of these AUG codons led to 3- to 5-fold increases in activity as measured by in vitro translation assays using capped mRNAs. RNA toeprint analysis demonstrated many stalled ribosomes flanking the AUG codons and secondary structures near the AUGs. Secondary structures may increase the ability of ribosomes to recognize the two AUGs, despite their poor initiation context. The degree of repression by uAUGs varied significantly depending on the cell lines tested, which may partly explain the differential tissue expression. Myf6 is a critical myogenic transcription factor with the striking feature of three tandem AUG codons at the translation initiation site. This structure reduced expression; removing two of these AUGs led to a doubling of activity in CV-1, HeLa, and NIH/3T3 cells.

3T3 Cells↗

PubMed1990

Alternative ribosomal initiation gives rise to chicken brain-type creatine kinase isoproteins with heterogeneous amino termini.

In higher eukaryotes three different types of creatine kinases (CK) are expressed: the muscle-specific M-CK, the ubiquitous cytoplasmic B-CKs, and the mitochondrial Mi-CKs. They fulfill multiple tasks in cells with an intensive energy metabolism. Isolated chicken B-CK can be resolved by two-dimensional gel electrophoresis into a major acidic Ba-CK and a major basic Bb-CK protein species which are very likely produced from the unique chicken B-CK gene (Wirz, T., Hossle, J. P., Soldati, T., and Perriard, J.-C. (1989) Experientia (Basel) 45, 32 (abstr.]. However, close inspection of the gels indicates the presence of additional B-CK species. This additional heterogeneity is generated by two distinct post-transcriptional processes. Post-translational phosphorylation was shown to contribute to heterogeneity of both Ba- and Bb-CK isoproteins and appears to modulate their enzymatic activity (A. F. Q. Quest, H. M. Eppenberger, and T. Wallimann, manuscript in preparation). Alternative ribosomal initiation of Bb-CK synthesis at multiple sites was shown to occur in cell free systems as well as in vivo, resulting in proteins differing in the length of their amino termini. Using site-directed mutagenesis to "switch off" each of the first four methionine codons of a full length Bb-CK cDNA, we were able to correlate each protein product with one distinct translational start site. An additional protein species appears to be produced by initiation at a noncanonical start codon. We propose that a leucine codon may be used as a translational start site. Evidence is presented to support the role of these amino-terminal truncated subunits in the regulation of the enzyme.

Amino Acid Sequence↗

PubMed1991

Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon.

The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the -3 position did not affect relative translation rates of the ADH protein at the second-instar larval stage but resulted in a 2.4-fold drop in translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region -1 to -9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.

Alcohol Dehydrogenase↗

PubMed1990

Structure of the beta-1,3-1,4-glucanase gene of Bacillus macerans: homologies to other beta-glucanases.

The nucleotide sequence of an 852 base pair (bp) DNA fragment containing the entire gene coding for thermostable beta-1,3-1,4-glucanase of Bacillus macerans has been determined. The bglM gene comprises an open reading frame (ORF) of 711 bp (237 codons) starting with ATG at position 93 and extending to the translational stop codon TAA at position 804. The deduced amino acid sequence of the mature protein shows 70% homology to published sequences of mesophilic beta-1,3-1,4-glucanases from B. subtilis and B. amyloliquefaciens. The sequence coding for mature beta-glucanase is preceded by a putative signal peptide of 25 amino acid residues, and a sequence resembling a ribosome-binding site (GGAGG) before the initiation codon. By contrast with the processed protein, the N-terminal amino acid sequence constituting the putative leader peptide bears no or only weak homology to signal peptides of mesophilic Bacillus endo-beta-glucanases. The B. macerans signal peptide appears to be functional in exporting the enzyme to the periplasm in E. coli. More than 50% of the whole glucanase activity was localized in the periplasmic space and in the supernatant. Whereas homology to endo-1,4-beta-glucanases is completely lacking, a weak amino acid homology between the sequence surrounding the active site of phage T4 lysozyme and a sequence spanning residues 126 through 161 of B. macerans endo-beta-glucanase could be identified.

Amino Acid Sequence↗

PubMed1982

Construction and characterization of E. coli promoter-probe plasmid vectors. III. pBR322 derivatives with deletions in the tetracycline resistance promoter region.

Deletions of the promoter region for the tetracycline-resistance (Tcr) gene(s) of pBR322 were constructed in order to generate new promoter-probe plasmid cloning vectors. The deletions were constructed in vitro by exonuclease digestion at the HindIII site and blunt-end ligation of the digestion products. Plasmids which lost the HindIII site but retained the EcoRI site carried deletions ranging from 5 to 60 bp. Some of the plasmids lacked the nucleotide sequences required for initiation of transcription from the Tcr promoter and "anti-Tcr" promoter. Three of the promoter-deletion plasmids (containing deletions of 5-29 bp) formed tight-binding complexes with RNA polymerase in vitro, despite their tetracycline sensitive phenotype. One deletion plasmid, pPV33, retained three out-of-phase stop codons located between the promoter-cloning site (EcoRI) and the translational start codon for the tetracycline resistance gene. These features give pPV33 several advantages over previously described promoter-cloning vehicles.

Cloning, Molecular↗

PubMed1996

Isolation and characterization of the Haemophilus influenzae tolQ, tolR, tolA and tolB genes.

The tolQ, R, A and B genes have been isolated from the DNA of Haemophilus influenzae and sequenced. The deduced amino acid (aa) sequence of the H. influenzae TolQ, TolR, TolA and TolB show 67, 63, 41 and 62% identity with Escherichia coli TolQRAB proteins, respectively. These four proteins are involved in transport of colicins and phages across the cell envelope. The translational stop codon of TolB (the last gene in the cluster) is 23 bases upstream of the start codon of the P6 lipoprotein gene. Primer extension and Northern blot analysis revealed that the start of the P6 transcript is within the tolB gene. Nucleotide sequence (nt) analysis of the entire tolQRABP6 region shows a transcriptional terminator immediately downstream of the P6 gene. The tolQRABP6 gene cluster of H. influenzae may thus constitute an operon.

Amino Acid Sequence↗

PubMed1999

Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis.

We analysed the Bacillus subtilis protein coding sequences termini, and compared it to other genomes. The analysis focused on signals, com-positional biases of nucleotides, oligonucleotides, codons and amino acids and mRNA secondary structure. AUG is the preferred start codon in all genomes, independent of their G+C content, and seems to induce less stable mRNA structures. However, it is not conserved between homologous genes neither is it preferred in highly expressed genes. In B.subtilis the ribosome binding site is very strong. We found that downstream boxes do not seem to exist either in Escherichia coli or in B.subtilis. UAA stop codon usage is correlated with the G+C content and is strongly selected in highly expressed genes. We found less stable mRNA structures at both termini, which we related to mRNA-ribosome and mRNA-release-factor interactions. This pattern seems to impose a peculiar A-rich nucleotide and codon usage bias in these regions. Finally the analysis of all proteins from B.subtilis revealed a similar amino acid bias near both termini of proteins consisting of over-representation of hydrophilic residues. This bias near the stop codon is partially release-factor specific.

Algorithms↗

PubMed1994

Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.

The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci.

Amino Acid Sequence↗

PubMed1999

Vector engineering anomalies: impact on fusion protein purification performance.

Recombinant protein purification using IMAC is often carried out by protein fusion to affinity tags. We have identified several tags useful for protein purification on Zn(II)-IDA columns. These tags were fused to the green fluorescent protein (rGFPuv) using the vector pGFPuv distributed by Clontech Lab (Palo Alto, CA) and analyzed for purification on Zn(II)-IDA. Each fusion protein exhibited elution heterogeneity (elution in two distinct pHs) from Zn(II)-IDA columns This led us to believe that two populations of fluorescent proteins were being expressed: one without the tag coeluting with Escherichia coli proteins at pH 7.5 and one bearing the tag eluting at a pH lower than pH 7.5. Assessment of the constructs revealed the possibility of a ribosomal binding site and start codon between the fusion tag and the rGFPuv sequence which might be used as a secondary translation start site. This hypothesis was confirmed by changing the second ATG (methionine) codon to an ACG (threonine) codon. The protein produced from this new construct eluted in a single fraction from a Zn(II)-IDA column. Thus, vector irregularities (along with other possibilities) should be examined when searching for the cause of elution heterogeneity of a target protein.

Chromatography, Affinity↗