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PubMed2002

Control of eukaryotic protein synthesis by upstream open reading frames in the 5'-untranslated region of an mRNA.

Control of gene expression is achieved at various levels. Translational control becomes crucial in the absence of transcription, such as occurs in early developmental stages. One of the initiating events in translation is that the 40 S subunit of the ribosome binds the mRNA at the 5'-cap structure and scans the 5'-untranslated region (5'-UTR) for AUG initiation codons. AUG codons upstream of the main open reading frame can induce formation of a translation-competent ribosome that may translate and (i) terminate and re-initiate, (ii) terminate and leave the mRNA, resulting in down-regulation of translation of the main open reading frame, or (iii) synthesize an N-terminally extended protein. In the present review we discuss how upstream AUGs can control the expression of the main open reading frame, and a comparison is made with other elements in the 5'-UTR that control mRNA translation, such as hairpins and internal ribosome entry sites. Recent data indicate the flexibility of controlling translation initiation, and how the mode of ribosome entry on the mRNA as well as the elements in the 5'-UTR can accurately regulate the amount of protein synthesized from a specific mRNA.

5' Untranslated Regions↗

PubMed2001

Functional cloning, based on azole resistance in Saccharomyces cerevisiae, and characterization of Rhizopus nigricans redox carriers that are differentially involved in the P450-dependent response to progesterone stress.

The filamentous fungus Rhizopus nigricans responds to treatment with progesterone by inducing P450-associated redox carriers. Selection for azole resistance following expression of a cDNA library constructed with RNA from progesterone-treated R. nigricans in the yeast Saccharomyces cerevisiae led to the identification of CPR1-FL and CYB5-1 cDNAs, which code for functionally competent NADPH-cytochrome P450 reductase and cytochrome b5, respectively. The central region (CPR2-CS) of an additional reductase gene sharing 66% identity with CPR1-FL was cloned from progesterone-induced mRNA by RT-PCR, using primers based on consensus sequences. Northern analysis of the 2.1-kb transcripts revealed that, of the two cloned reductase genes, only CPR1-FL mRNA was strongly induced by progesterone; transcription of CYBS-1 and CPR2-CS mRNAs was not significantly affected. Analysis of the subcellular localization and function of the R. nigricans reductase in yeast indicated that the CPR1-FL cDNA and a derivative (CPR1-S) truncated at the first ATG codon gave rise to functionally equivalent products that were found in both cytosolic and microsomal fractions. In contrast, addition of an in-frame initiation codon at the 5' end of the CPR1-FL sequence resulted in localization of the activity mainly to the microsomes, and improved ketoconazole resistance but decreased NADPH-cytochrome c reductase activity in the host strain. These findings suggest that, of the three genes for P450-associated redox carriers investigated, only CPR1-FL is associated with the progesterone response and that its major transcript encodes a reductase that shows an unusual pattern of subcellular localization.

Amino Acid Sequence↗

PubMed1999

Identification and characterization of a germination operon on the virulence plasmid pXO1 of Bacillus anthracis.

The spores of Bacillus anthracis, the agent of anthrax disease, germinate within professional phagocytes, such as murine macrophage-like RAW264.7 cells and alveolar macrophages. We identified a cluster of germination genes extending for 3608 nucleotides between the pag and atxA genes on the B. anthracis virulence plasmid pXO1. The three predicted proteins (40, 55 and 37 kDa in size) have significant sequence similarities to B. subtilis, B. cereus and B. megaterium germination proteins. Northern blot analysis of total RNA from sporulating cells indicated that the gerX locus was organized as a tricistronic operon (gerXB, gerXA and gerXC). Primer extension analysis identified a major potential transcriptional start site 31 bp upstream from the translation initiation codon of gerXB. Expression of the gerX operon was studied using a gerXB-lacZ transcriptional fusion. Expression began 2.5-3 h after the initiation of sporulation and was detected exclusively in the forespore compartment. A gerX null mutant was constructed. It was less virulent than the parental strain and did not germinate efficiently in vivo or in vitro within phagocytic cells. These data strongly suggest that gerX-encoded proteins are involved in the virulence of B. anthracis.

Animals↗

PubMed1982

Structure-function relationship of Rous sarcoma virus leader RNA.

Cells infected by RSV synthesize viral 35S RNA as well as subgenomic 28S and 22S RNAs coding for the Env and Src genes respectively. In addition, at least the 5' 101 nucleotides of the leader are also conserved and we have shown previously that this sequence contains a strong ribosome binding site (J.-L. Darlix et al., J. Virol. 29, 597). We now report the RNA sequence of Rous Sarcoma virus (RSV) leader RNA and propose a folding of this 5' untranslated region which brings the Cap, the initiation codon for Gag and the strong ribosome binding site close to each other. We also show that ribosomes protect a sequence just upstream from initiator Aug of Gag in vitro, and believed to interact with part of the strong ribosome binding site according to the folding proposed for the leader RNA.

Animals↗

PubMed1978

Nucleotide sequence at the 5' end of ovalbumin messenger RNA from chicken.

DNA-sequence analysis of 300 nucleotides from the region of cloned, double-stranded ovalbumin cDNA corresponding to the 5' end of ovalbumin messenger RNA was accomplished using the technique of Maxam and Gilbert (Proc. Nat. Acad. Sci. USA (1977) 74,560-564). The AUG initiation codon was located 52 nucleotides from the AT linkers used in cloning and immediately adjacent to the amino terminal peptide of ovalbumin, indicating the absence of a "signal peptide" in this protein. The nucleotide sequence coding for a phosphorylated peptide from ovalbumin was also found. These results demonstrate that the coding portion of mRNAov begins near the 5' end of the molecule leaving some 600 nucleotides of noncoding information at the 3' end.

Animals↗

PubMed2003

Isolation and characterization of the neutral leucine aminopeptidase (LapN) of tomato.

Tomatoes (Lycopersicon esculentum) express two forms of leucine aminopeptidase (LAP-A and LAP-N) and two LAP-like proteins. The relatedness of LAP-N and LAP-A was determined using affinity-purified antibodies to four LAP-A protein domains. Antibodies to epitopes in the most N-terminal region were able to discriminate between LAP-A and LAP-N, whereas antibodies recognizing central and COOH-terminal regions recognized both LAP polypeptides. Two-dimensional immunoblots showed that LAP-N and the LAP-like proteins were detected in all vegetative (leaves, stems, roots, and cotyledons) and reproductive (pistils, sepals, petals, stamens, and floral buds) organs examined, whereas LAP-A exhibited a distinct expression program. LapN was a single-copy gene encoding a rare-class transcript. A full-length LapN cDNA clone was isolated, and the deduced sequence had 77% peptide sequence identity with the wound-induced LAP-A. Comparison of LAP-N with other plant LAPs identified 28 signature residues that classified LAP proteins as LAP-N or LAP-A like. Overexpression of a His(6)-LAP-N fusion protein in Escherichia coli demonstrated distinct differences in His(6)-LAP-N and His(6)-LAP-A activities. Similar to LapA, the LapN RNA encoded a precursor protein with a molecular mass of 60 kD. The 5-kD presequence had features similar to plastid transit peptides, and processing of the LAP-N presequence could generate the mature 55-kD LAP-N. Unlike LapA, the LapN transcript contained a second in-frame ATG, and utilization of this potential initiation codon would yield a 55-kD LAP-N protein. The localization of LAP-N could be controlled by the balance of translational initiation site utilization and LAP-N preprotein processing.

Amino Acid Sequence↗

PubMed1992

The luxR gene product of Vibrio harveyi is a transcriptional activator of the lux promoter.

Expression of the lux operon from the marine bacterium Vibrio harveyi is dependent on cell density and requires an unlinked regulatory gene, luxR, and other cofactors for autoregulation. Escherichia coli transformed with the lux operon emits very low levels of light, and this deficiency can be partially alleviated by coexpression of luxR in trans. The V. harveyi lux promoter was analyzed in vivo by primer extension mapping to examine the function of luxR. RNA isolated from E. coli transformed with the Vibrio harveyi lux operon was shown to have a start site at 123 bp upstream of the first ATG codon of luxC. This is in sharp contrast to the start site found for lux RNA isolated from V. harveyi, at 26 bp upstream of the luxC initiation codon. However, when E. coli was cotransformed with both the lux operon and luxR, the start site of the lux mRNA shifted from -123 to -26. Furthermore, expression of the luxR gene caused a 350-fold increase in lux mRNA levels. The results suggest that LuxR of V. harveyi is a transcriptional activator stimulating initiation at the -26 lux promoter.

Bacterial Proteins↗

PubMed1996

Studies on the Expression of Recombinant Human MCAF/GM-CSF Fusion Protein and Its Biological Activities.

With PCR technology and methods of DNA recombination in vitro, the fusion protein of GM-CSF with MCAF, the facto that has a directing effect on cells to a target, was generated by the construction of recombinant plasmids pMG 01, pMG 02 and pMG 03 with different nucleotide composition between its SD sequence and the initiation codon ATG under the control of lambdaP(R) P(L) promoter of vector pBV 220. Although no stable secondary structure exist in the translation initiation regions of all the recombinant plasmid constructed, the expression levels of the products with DH5 alpha (pMG 02) and DH5alpha (pMG 03) are much higher than that with DH5alpha (pMG01) which hardly expressed the product. The assay of Western blot indicated that the expressed product reacted with MCAF and GM-CSF antibodies, respectively. The assay of biological activities showed that the expressed product had apparent monocyte chemoattractant activity and supported the growth of the human GM-CSF-dependent cell line TF1, suggesting that the biological functions of MCAF and GM-CSF are compatible

Journal Article↗

PubMedDate not supplied

[Correlation between the effectiveness of translation initiation and secondary structure of mRNA in the hybrid gene cro-lacIZ].

A set of plasmids containing short DNA deletions in the N-part of cro-lacIZ coding zone as constructed using Bal31 nuclease. Development of models of mRNA secondary structure was carried out stepwise beginning from the 5'-end by taking into consideration the hairpins first formed during mRNA synthesis. Comparison of the results of mRNA secondary structure determination and protein production analysis demonstrated a correlation between the efficiency of translation initiation and the appearance of a single-stranded region upon disruption of the mRNA local secondary structure in the translation initiation zone generated by ribosomes. These results confirm the suggestion of the central role played by the Shine-Dalgarno sequence in the generation of a single-stranded region. Some plasmids from the set are supposed to determine protein synthesis by a translation reinitiation mechanism in the absence of Shine--Dalgarno interaction. In this case, correlation between reinitiation efficiency and local disruption of the mRNA secondary structure by the terminating ribosome was also observed. The terminating ribosome that forms the single-stranded region near the initiation codon fulfils the major function of the Shine--Dalgarno interaction. In addition, the possible effect of another mRNA secondary structure region on the translation initiation efficiency is discussed.

Escherichia coli↗

PubMed1986

Quantitative analysis of the relationship between nucleotide sequence and functional activity.

Matrices can be used to evaluate sequences for functional activity. Multiple regression can solve for the matrix that gives the best fit between sequence evaluations and quantitative activities. This analysis shows that the best model for context effects on suppression by su2 involves primarily the two nucleotides 3' to the amber codon, and that their contributions are independent and additive. Context effects on 2AP mutagenesis also involve the two nucleotides 3' to the 2AP insertion, but their effects are not independent. In a construct for producing beta-galactosidase, the effects on translational yields of the tri-nucleotide 5' to the initiation codon are dependent on the entire triplet. Models based on these quantitative results are presented for each of the examples.

Base Sequence↗

PubMed1993

Gene structure and expression of rice seed allergenic proteins belonging to the alpha-amylase/trypsin inhibitor family.

Genomic and two novel cDNA clones for rice seed allergenic protein (RA) belonging to the alpha-amylase/trypsin inhibitor family were isolated and their nucleotide sequences determined. Ten cysteine residues deduced from nucleotide sequences were completely conserved among three cDNA clones including a clone, RA17, reported previously. One genomic clone, lambda 4, contained two RA genes, RAG1 and RAG2. Although RAG1 was cloned at the 5' portion only, two RA genes were arranged divergently. Nucleotide sequencing and DNA blotting analyses showed that RA are encoded by a multigene family consisting of at least four members. The transcriptional initiation site of RAG1 was localized at A, 26 bp upstream of the putative translational initiation codon, ATG, by the primer extension assay. The putative TATA box and CAAT box existed about 45 bp and 147 bp upstream of the transcription initiation site, respectively. A conserved sequence (ATGCAAAA) which was similar to the sequence (TGCAAAA) identified in rice glutelin promoters was observed in the 5' region of the two genes. In addition, RNA blotting analyses provided that RA genes specifically expressed in ripening seed and their transcripts accumulated maximally between 15 and 20 days after flowering.

Allergens↗

PubMed1995

Cloning and characterization of the promoter region of the mouse mu opioid receptor gene.

Opioid compounds have potent analgesic and euphoric properties. They act with specific cell-membrane receptors which have been pharmacologically defined into three major classes, mu, kappa and delta. These receptors are highly regulated with respect to their gene expression, resulting in a temporally and spatially specific pattern of distribution for each receptor. To characterize the promoter sequence of the mu opioid receptor (MOR) gene, a mouse genomic DNA library was screened under high stringency with a rat MOR (MOR-1) cDNA probe and genomic sequences for the mouse MOR gene were isolated. From one genomic clone, a 2.3-kb EcoRI fragment, which hybridized to the 5'-end of the rat MOR-1 cDNA probe, was subcloned and sequenced. This fragment contains 1.3 kb of sequence upstream of the initiation codon, extends downstream through exon 1 and includes a portion of intron 1. Primer extension analysis using mouse brain poly (A)+ RNA identified a transcription initiation site 793 bp upstream from the translation start site. Chimeric constructs of mouse MOR deletion fragments fused to a luciferase reporter gene were transfected into a human neuroblastoma cell line, SK-N-SH, which constitutively expresses endogenous MOR. These transient expression studies indicated that the 0.2-kb region upstream from the transcription initiation site possesses a functional promoter, which directs the expression of the reporter gene in vitro and may possess promoter activity for the mouse MOR gene in vivo.

Animals↗

PubMed1986

In vitro transcription and translation of cloned cDNAs encoding the 30-kDa protein gene of TMV.

A cDNA clone encoding the nonstructural, 30-kDa protein of the common (U1) strain of tobacco mosaic virus (TMV) was isolated and characterized. cDNA clones representing the intact gene as well as deletions from the 5' end of the gene were subcloned into SP6 vectors. Capped RNAs produced by in vitro transcription reactions were translated in a wheat germ cell-free system. The resultant proteins were compared to proteins obtained from the in vitro translation of intermediate length (I2) rods of TMV. Transcripts of the cDNA clones encoded polypeptides of 30, 28, or 18 kDa that were immunoprecipitated by antibody prepared against a synthetic peptide representing the carboxy terminus of the 30-kDa protein. cDNA clones containing the intact 30-kDa sequence coded for 30-kDa polypeptides while clones lacking the 30-kDa initiation codon produced 28-kDa polypeptides. Surprisingly, translation of a transcript from a cDNA clone containing the 30-kDa gene plus 390 nucleotides 5' of the initiator AUG yielded a polypeptide with an approximate molecular mass of 18 kDa. The results indicate that an intact and functional 30-kDa protein gene has been cloned. The significance of these results, with respect to determining the function of the 30-kDa protein, is discussed.

Cell-Free System↗

PubMed2001

Molecular cloning of the orphanin FQ receptor gene and differential tissue expression of splice variants in rat.

Pharmacological and receptor-ligand binding studies of the cloned orphanin FQ (OFQ) receptor suggest that multiple forms of this receptor may exist. To further characterize the OFQ receptor (OFQR), we attempted to isolate the gene encoding this receptor in rat. The OFQR gene exceeds 10 kb in length and contains six exons ranging from 34 to 524 bp that are interrupted by five introns. The ATG translation initiation codon is located in exon 2, and the open reading frame consists of 1283 bp. Primer extension analysis of the gene revealed two major transcription initiation sites: one in the 5' flanking region and the other in intron 1. The rat OFQR gene appeared to be alternatively spliced to yield multiple mRNAs. Four splice variants deleted for exon 1 were expressed only in brain. In contrast, five isoforms containing exon 1 were expressed in various tissues, such as brain, testes, and gastrointestinal tract. These data suggest that unique regions in intron 1 and in the 5' flanking region of the OFQR gene contribute to the regulation of its expression in different tissues.

Alternative Splicing↗

PubMed1998

The gene structure and promoter sequence of mouse hyaluronan synthase 1.

The structure and organization of mouse hyaluronan synthase 1 gene, HAS1 were determined by direct sequencing of lambda phage clones carrying the entire gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon-intron boundaries and all of the exons. This gene spans about 11kb of genomic DNA and consists of 5 exons and 4 introns. A similarity in the exon-intron organization was found between the genes of mouse HAS1 and Xenopus laevis DG42 which was recently identified as Xenopus hyaluronan synthase. The transcription initiation site was determined by rapid amplification of the cDNA ends (5'-RACE). Position +1 is located 55 nucleotides upstream of the ATG initiation codon. The promoter region of the HAS1 gene has no typical TATA box, but contains a CCAAT box located 190 nucleotides upstream of the transcription initiation site. Further analysis of 1.4 kb of the 5' flanking region revealed several potential binding motifs for transcription factors. This information about the gene structure may be useful for further studies on the promoter activity.

Animals↗

PubMed1997

Identification of the human alpha6 integrin gene promoter.

The alpha6 integrin subunit couples with either the beta1 or the beta4 subunit to form a laminin receptor. alpha6 expression is cell-type-specific and generally is present at high levels in epithelial and endothelial cells. To study its gene regulation, we isolated a genomic clone containing the human alpha6 integrin gene promoter. It includes 3 kb of the upstream flanking region, the first exon (385 bp), and 9 kb of the first intron. The alpha6 promoter directs transcription initiation from a primary site 202 nucleotides from the translation initiation codon. Unlike most other integrin gene promoters, the alpha6 promoter has a TATA box (GATAAA), which is located 22 nucleotides upstream from the primary transcription initiation site. A 190-bp region upstream from the TATA box is highly rich (78%) in C and G nucleotides and contains several Sp1 and AP2 binding sequences. However, full promoter activity (in the presence of the SV40 enhancer) requires only 78 bp of this C/G-rich sequence upstream from the TATA box. Slightly upstream from the C/G-rich region are a steroid receptor binding homolog and an epithelial-cell-specific E-pal sequence. Another possible epithelial cell-specific binding sequence (Ker1) is found immediately downstream from the TATA box. Cell-type-specific activities of the promoter paralleled the alpha6 mRNA levels in four tested cell lines. In the presence of the SV40 enhancer, alpha6 promoter activity increased approximately four-fold in primary keratinocytes and in HT1080 fibrosarcoma cells and 30-fold in T47D breast carcinoma cells, but remained undetectable in K562 leukemia cells. Genomic analysis that compared alpha6-expressing with non-alpha6-expressing cells suggested that DNA methylation is not involved in the silencing of the alpha6 gene in alpha6-negative cells. DNase I footprint analysis confirmed the binding of Sp1 and AP2 to their cognate sequences. A nuclear extract of high-alpha6-expressing HBL-100 cells also produced significant binding to these sites, suggesting that the two transcription factors are probably involved in the positive regulation of the alpha6 promoter.

Amino Acid Sequence↗

PubMed1998

Gene and cDNA structures of flounder insulin-like growth factor-I (IGF-I): multiple mRNA species encode a single short mature IGF-I.

To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus. The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb. By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486. In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724. The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression.

Amino Acid Sequence↗

PubMed1993

cis-acting elements in the 5'-untranslated region of rat testis proenkephalin mRNA regulate translation of the precursor protein.

We found that a rat testis cDNA library contained two types of proenkephalin cDNA, corresponding to somatic and germ cell-specific transcripts. The somatic form is identical in primary sequence to previously characterized transcripts from rat brain and heart. The longer germ cell-specific cDNA contains a novel 5' terminus derived from the somatic intron A. We found that germ cell-specific transcripts translate in vitro with significantly lower efficiency than do somatic-type transcripts. Deletion analysis indicated that translational inefficiency is mediated primarily by the presence of four upstream AUG codons followed by short open reading frames within the leader sequence. Secondary structure in the form of a stem-and-loop upstream of the initiator codon for proenkephalin was found to have a slightly inhibitory effect on translation. These results, coupled with the very low abundance of the somatic transcript, provide a possible explanation for the low levels of opioid peptides found in adult rat testis in spite of the abundant quantity of germ cell-specific proenkephalin transcript.

Animals↗