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PubMed1992

The effect of ribosomal protein S1 from Escherichia coli and Micrococcus luteus on protein synthesis in vitro by E. coli and Bacillus subtilis.

We have designed a set of nine plasmids containing the Bacillus pumilis cat gene with one of three Shine-Dalgarno (SD) sequences (weak, strong or stronger) and one of three initiation codons (AUG, GUG or UUG). These constructions have been used to determine the effect of ribosomal protein S1, SD and initiation codon sequences and Escherichia coli ribosomal protein S1 on translation in vitro by E. coli and B. subtilis ribosomes. Translation of these nine constructions was determined with three types of ribosomes: E. coli containing ribosomal protein S1, E. coli depleted of S1, and B. subtilis which is naturally free of S1. E. coli ribosomes were able to translate all nine transcripts with variable efficiencies. B. subtilis and S1-depleted E. coli ribosomes were similar to each other and differed from non-depleted E. coli ribosomes in that they required strong or stronger SD sequences and were unable to translate any of the weak transcripts. Addition of S1 from either E. coli or Micrococcus luteus, a Gram-positive bacterium, enabled S1-depleted E. coli ribosomes to translate mRNAs with weak SD sequences but had no effect on B. subtilis ribosomes. AUG was the preferred initiation codon for all ribosome types; however, B. subtilis ribosomes showed greater tolerance for the non-AUG codons than either type of E. coli ribosome. The presence of a strong or stronger SD sequence increased the efficiency by which E. coli ribosomes could utilize non-AUG codons.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus subtilis↗

PubMed1991

Cloning and sequencing of a cyclodextrin glucanotransferase gene from Bacillus ohbensis and its expression in Escherichia coli.

A cyclodextrin glucanotransferase (CGTase) gene of Bacillus ohbensis was cloned in Escherichia coli and the nucleotide sequence was determined. A single open reading frame (2112 bp) with a TTG codon as an initiator was identified that encodes a typical signal peptide of 29 amino acids followed by the mature enzyme (675 amino acids), of which the partial amino acid sequences of the N-terminal region and some lysyl-endopeptidase fragments were determined by Edman degradation. The CGTase gene was expressed in E. coli under control of the lac promoter only when the upstream region containing a long inverted repeat structure (located at -108 to -67 bp from the initiation codon) was deleted. Substitution of an ATG codon for the initiation TTG triplet doubled the expression of the CGTase gene in E. coli. Enzyme preparations purified from the culture supernatant of B. ohbensis and from the periplasmic fraction of the E. coli transformant exhibited the same molecular weight (Mr) and enzymatic properties as follows: Mr, 80,000; optimum pH for activity, 5.0 (and a suboptimum at 10.0); stability between pH 6.5 and 10.0; optimum temperature for activity, 55 degrees C; and stability below 45 degrees C. The yields of the products from starch as the substrate were 25% for beta- and 5% for gamma-cyclodextrin.

Amino Acid Sequence↗

PubMed1993

Nucleotide sequence analysis of the precore region in patients with fulminant hepatitis B in the United States.

BACKGROUND: A precore defective hepatitis B virus (HBV) mutant unable to produce hepatitis B e antigen (HBeAg) has been associated with fulminant hepatitis B. We have studied the etiologic contribution of precore mutants among North American patients with this disorder. METHODS: We studied 39 patients with fulminant hepatitis B. The precore and proximal core regions of HBV from 37 of 39 patients were sequenced. RESULTS: Four patients (10.8%) harbored nonsense mutants likely to produce an HBeAg negative HBV infection; two such mutants had a G to A substitution at position 1896, one lost the precore initiation codon, and one harbored a stop codon immediately downstream of the precore initiation codon. Recovered sequences from seven additional patients displayed silent or missense mutations in these regions. All delta coinfected patients harbored known wild type strains of HBV. A significantly poorer survival was associated with antibody to HBe positivity and presence of nucleotide substitutions in the precore/core region. CONCLUSIONS: The prevalence of precore mutations in 37 patients from the United States was lower than reported elsewhere; only two patients were found to have the G to A transition mutation in the precore region at position 1896. We conclude that HBeAg negative HBV mutants do not play a predominant etiologic role among North American patients with fulminant hepatitis B.

Adenine↗

PubMed2004

Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator.

The Sm-like protein Hfq is involved in post-transcriptional regulation by small, noncoding RNAs in Escherichia coli that act by base pairing. Hfq stabilises the small RNAs and mediates their interaction with the target mRNA by an as yet unknown mechanism. We show here a novel chaperoning use of Hfq in the regulation by small RNAs. We analysed in vitro and in vivo the role of Hfq in the interaction between the small RNA RyhB and its sodB (iron superoxide dismutase) mRNA target. Hfq bound strongly to sodB mRNA and altered the structure of the mRNA, partially opening a loop. This gives access to a sequence complementary to RyhB and encompassing the translation initiation codon. RyhB binding blocked the translation initiation codon of sodB and triggered the degradation of both RyhB and sodB mRNA. Thus, Hfq is a critical chaperone in vivo and in vitro, changing the folding of the target mRNA to make it subject to the small RNA regulator.

Bacterial Proteins↗

PubMed1985

Molecular structure of the human cytoplasmic beta-actin gene: interspecies homology of sequences in the introns.

A recombinant phage that carries the cytoplasmic beta-actin gene was isolated from a human DNA library. The nucleotide sequence of this gene was determined. The amino acid sequence deduced from the nucleotide sequence matches perfectly that of beta-actin from human fibroblasts. The gene contains five introns. A large intron was found in the 5' untranslated region six nucleotides upstream from the ATG initiation codon. Four introns were found within the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. In contrast to the human cardiac muscle actin gene, the aorta-type smooth muscle actin gene, and the stomach-type smooth muscle actin gene, the beta-actin gene lacks the codon for cysteine between the ATG initiation codon and the codon for the NH2-terminal amino acid of the mature protein. Hybridization of genomic DNA with DNA fragments derived from intron I in the 5' untranslated region and from intron III strongly suggests the presence of a single beta-actin gene in the human genome. The DNA sequences of the coding region, of the 3' untranslated region, and of the sequence block between the "CCAAT" box and "TATA" box in the 5' flanking DNA of the human beta-actin gene are highly homologous to the corresponding sequences of the rat and chicken beta-actin genes. Unexpectedly, the sequence of intron III of the human beta-actin gene shows considerable homology to that of the rat beta-actin gene.

Actins↗

PubMed1994

An efficient Shine-Dalgarno sequence but not translation is necessary for lacZ mRNA stability in Escherichia coli.

The 5' ends of many bacterial transcripts are important in determining mRNA stability. A series of Shine-Dalgarno (SD) sequence changes showed that the complementarity of the SD sequence to the anti-SD sequence of 16S rRNA correlates with lacZ mRNA stability in Escherichia coli. Several initiation codon changes showed that an efficient initiation codon is not necessary to maintain lacZ mRNA stability. A stop codon in the 10th codon of lacZ increased mRNA stability. Therefore, ribosomal binding via the SD sequence but not translation of the coding region is necessary to maintain lacZ mRNA stability.

Codon↗

PubMed1999

The two forms of karyogamy transcription factor Kar4p are regulated by differential initiation of transcription, translation, and protein turnover.

Kar4p is a transcription factor in Saccharomyces cerevisiae that is required for the expression of karyogamy-specific genes during mating, for the efficient transit from G1 during mitosis, and for essential functions during meiosis. Kar4p exists in two forms: a constitutive slower-migrating form, which predominates during vegetative growth, and a faster-migrating form, which is highly induced by mating pheromone. Transcript mapping of KAR4 revealed that the constitutive mRNA was initiated upstream of two in-frame ATG initiation codons, while the major inducible mRNA originated between them. Thus, the two forms of Kar4p are derived from the translation of alternative transcripts, which possess different AUG initiation codons. Site-directed mutations were constructed to inactivate one or the other of the initiation codons, allowing the expression of the two Kar4p forms separately. At normal levels of expression, the constitutive form of Kar4p did not support wild-type levels of mating. However, the two forms of Kar4p could also be expressed separately from the regulatable GAL1 promoter, and no functional difference was detected when they were expressed at equivalent levels. Pulse-chase experiments showed that the induced form of Kar4p was highly expressed and stable during mating but rapidly turned over in vegetative cells. In contrast, the constitutively expressed longer form showed the same rate of turnover regardless of the growth condition. Furthermore, overexpression of either form of Kar4p in vegetative cells was toxic. Thus, the elaborate regulation of the two forms of Kar4p at the levels of transcription, translation, and protein turnover reflects the requirement for high levels of the protein during mating and for low levels during the subsequent phases of the cell cycle.

Chromosome Mapping↗

PubMed1984

Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs.

5-Noncoding sequences have been tabulated for 211 messenger RNAs from higher eukaryotic cells. The 5'-proximal AUG triplet serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine (most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine in that position. There is a predominance of C in positions -1, -2, -4 and -5, just upstream from the initiator codon. The sequence CCAGCCAUG (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in a given mRNA matches the -1 to -5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides in common with the CCACC consensus, but only ten mRNAs conform perfectly.

Base Sequence↗

PubMed1984

Affecting gene expression by altering the length and sequence of the 5' leader.

We have utilized a recombinant vector system designed to study gene control elements by fusing them to the Escherichia coli galactokinase gene (galK) to examine the effects of gene expression that result from introducing changes into the 5' noncoding leader region of the galK transcription unit. We varied the length of the 5' leader region and found no effect on galK expression, provided that the galK initiation codon remained the first AUG in the transcription unit. Using synthetic linkers, we then inserted specific sequences, each containing a single AUG codon at a defined position within the leader region. We found that the AUG inserts had widely different effects on galK expression and that the sequences surrounding the inserted AUG codons determined the magnitude of these effects. In addition, we placed these upstream inserted AUG codons into each of the three possible translation reading frames so that translation occurring in these frames terminates prior to, within, or downstream of the galK initiation codon, respectively. Single-nucleotide frameshift mutations were also introduced into one of these constructs to shift upstream translation into the other two possible reading frames. Depending upon where upstream translation stopped relative to the galK initiation codon, we observed consistently different effects on galK expression. Our results show that an upstream AUG that interferes with downstream translation initiation exerts its greatest effect when it translates out-of-frame through the downstream initiator into the gene. If translation is stopped upstream of or within the initiator, an unexpectedly high level of expression from the downstream AUG is maintained.

Codon↗

PubMed2000

Negative and translation termination-dependent positive control of FLI-1 protein synthesis by conserved overlapping 5' upstream open reading frames in Fli-1 mRNA.

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5' upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG -41 and GUG -37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5' uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5' uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5' uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons.

3T3 Cells↗

PubMed1989

Modulation of ras expression by anti-sense, nonionic deoxyoligonucleotide analogs.

Anti-ras oligodeoxyribonucleoside methylphosphonates (ONMP's) complementary to the initiation codon region have been synthesized to explore their efficacy and specificity on ras-p21 translation. ONMP (IC-0) precisely complementing the first initial 11 nucleotides of the Balb-ras initiation codon region acts in a dose-dependent manner to inhibit p21 translation by a rabbit reticulocyte lysate. At 100 microM, IC-0 inhibits the cell-free translation of p21 close to completion. The two control oligomers containing one or two nucleotide mismatches were significantly less effective than IC-0 at the equivalent concentration. In living cells, a perfectly matched ONMP directed against the initiation codon region inhibited Ha-ras p21 expression by 90% at a concentration of 50 microM.

Animals↗

PubMed1996

The three dominant female-sterile mutations of the Drosophila ovo gene are point mutations that create new translation-initiator AUG codons.

The Drosophila ovo gene, which encodes a putative transcription factor (Ovo) with TFIIIA-like zinc fingers, is required for female germline survival and proper oogenesis. Three dominant female-sterile ovoD mutations cause ovarian abnormalities that define an allelic series, with ovoD1 displaying the stronger phenotype and ovoD3 the weaker. We report here that all three ovoD mutations are point mutations that create new in-frame methionine codons in the 5' part of ovo. There are two types of overlapping ovo transcription units, ovo alpha and ovo beta. By using various ovo-lacZ reporter genes, we determined that the long Ovo isoforms starting at methionine M1, present in transcripts ovo alpha, are expressed at low levels only in mature oocytes. Short Ovo isoforms are translated from methionine M373, the first in-frame start codon present in transcript ovo beta, and correspond to the activity defined by recessive loss of function ovo mutations. The new AUGs created in ovoD mutations all are located upstream of the M373 initiation site. Our results support the hypothesis that they can substitute for M373 as translation starts and initiate the synthesis of Ovo proteins that have extra amino acids at their N termini. We propose that premature expression of long Ovo protein isoforms occurs in ovoD mutants and interferes with wild-type Ovo function in controlling female germline differentiation.

Amino Acid Sequence↗

PubMed1992

Expression in vivo and in vitro of the major structural protein (VP73) of African swine fever virus.

The VP73 protein was produced by in vitro transcription and translation from the Xho I-Bam HI fragment located between the Cla I-N and Cla I-H fragments of the viral genome. This DNA fragment encodes a late mRNA of about 2.6 kb detected in infected MS monkey and BHK hamster cells. The transcript was initiated at a site within two bases upstream of the translation initiation codon. The in vitro synthesized polypeptide shows the same molecular weight as the in vivo synthesized polypeptide, suggesting that VP73 has no post-translational modification. There are two internal AUG initiation codons for in vitro translation, one of which is functional in vivo, as well as a possible GUG initiator codon detected by expression of the protein in E. coli cells.

African Swine Fever Virus↗

PubMed1993

Biosynthesis of reovirus-specified polypeptides. Analysis of ribosome pausing during translation of reovirus S1 and S4 mRNAs in virus-infected and vector-transfected cells.

The steady-state distribution of translating ribosomes on the reovirus s1 and s4 mRNAs was determined using a sensitive primer extension inhibition assay and cDNA clones of the reovirus S1 and S4 genes. Positions of significant ribosome pausing included the sites of initiation and termination of translation, both in vivo in reovirus-infected mouse fibroblast L cells and in vitro in rabbit reticulocyte lysates. The patterns of ribosome pausing detected in vivo and in vitro were comparable. Ribosomes were far less uniformly distributed along the polycistronic s1 mRNA than they were along the monocistronic s4 mRNA. During reovirus infection, ribosome pausing was detected at the initiation codons of the s1 mRNA sigma 1 and sigma 1ns open reading frames and the s4 mRNA sigma 3 open reading frame, direct evidence that initiation is a slow step of translation in vivo. Quantitative analysis of reovirus mRNA translation in infected and transfected cells suggested that ribosomes paused longer at an AUG initiation codon present in strong context than one present in weak context. These results suggest that the dynamics of ribosome movement during translation of the reovirus s1 and s4 mRNAs are similar in vivo and in vitro and that the relative ribosome pause at an AUG initiation codon may be affected by the flanking nucleotide context of the AUG codon.

Animals↗

PubMed1984

S1 nuclease mapping of viral RNAs from a temperature-sensitive transformation mutant of murine sarcoma virus.

The structures of murine sarcoma virus (MuSV) ts110 viral RNA and intracellular RNA present in MuSV ts110-infected cells (6m2 cells) have been examined by S1 nuclease analysis. A previous study involving heteroduplex analysis of MuSV ts110 viral RNAs hybridized to wild-type DNA revealed the presence of two MuSV ts110 RNAs, 4.0 and 3.5 kilobases (kb) in length, containing overlapping central deletions relative to wild-type MuSV 124 viral RNA (Junghans et al., J. Mol. Biol. 161:229-255, 1982). Here we show that the deletion (termed delta 1) in the 4.0-kb RNA has a 5' border located at about nucleotide 2409 (using the numbering system of Van Beveren et al., Cell 27:97-108, 1981), a position 63 bases upstream of the junction of the p30 and p10 coding sequences. The 3' border of the delta 1 deletion is found 1,473 bases downstream at approximately nucleotide 3883, 10 nucleotides downstream of the first mos gene initiation codon. In the 3.5-kb MuSV ts110 RNA, the 5' border of the deleted central region (termed delta 2) is located in a splice consensus donor site at approximately nucleotide 2017, 330 bases downstream from the junction of the p12 and p30 coding sequences, and extends about 1,915 bases in the downstream direction to nucleotide 3935, found in a splice consensus acceptor site about 55 nucleotides downstream of the first mos gene initiation codon and 30 bases upstream of the second initiation codon. No alteration of polyadenylate addition sites was observed in either MuSV ts110 RNA species, as compared with MuSV 349 RNA. The observation that the 5' and 3' borders of the deletion in the 3.5-kb RNA are within in-frame splice donor and acceptor sites suggests strongly that the 3.5-kb RNA is derived from the 4.0-kb RNA by a temperature-sensitive splice mechanism. Data presented here show unequivocally that formation of the 3.5-kb MuSV ts110 RNA from which the P85gag-mos polypeptide is translated is temperature sensitive. At 33 degrees C, with S1 analysis, the 3.5-kb RNA is found readily in 6m2 cells. Within 4 h of a shift to 39 degrees C, however, only trace amounts of this RNA can be found. Moreover, reshifting 6m2 cells to 33 degrees C permits the reappearance of the 3.5-kb RNA at its original level.

Animals↗

PubMed1986

Evidence that AGUAUAUGA and CCAAGAUGA initiate translation in the same mRNA region E3 of adenovirus.

We described a simple method to introduce site-specific mutations into region E3 of adenovirus (Ad). Mutations are made in cloned Ad2 EcoRI-D (map position 76-83), then ligated between Ad5 EcoRI-A (map position 0-76) and EcoRI-B (map position 83-100) to complete the viral genome. We have used this method to isolate a viable virus mutant (dl702) that is relevant to the problems of translation initiation and gene organization in the E3 complex transcription unit. mRNA a in region E3 encodes an abundant glycoprotein termed gp19K. There are two AUGs in mRNA a that are 5' to AUG1204 which initiates gp19K. One of these, AUG1022, could initiate a 6.7K protein, although this protein has not been identified in infected cells. Mutant dl702 has a deletion such that the 6.7K gene is fused in-frame to the gp19K gene. We report that the 6.7K-gp19K fusion protein is synthesized both in dl702-infected cells and after cell free translation of infected cell RNA. The quantity of fusion protein made is much less than that of wild type gp19K. The sequence context of AUG1022 for 6.7K is AGUAUAUGA, and that of AUG1204 for gp19K is CCAAGAUGA. The consensus sequence of eukaryotic initiation codons is CCPuCCAUGG, with the Pu at -3 being important (M. Kozak, Nucleic Acids Res. 12, 857-872, 1984). Our results suggest that (i) AUG1022 can initiate translation in vivo and therefore the 6.7K protein probably is made in infected cells, (ii) that mRNA a is a dicistronic mRNA encoding the 6.7K and gp19K proteins, and (iii) that the initiation codon for 6.7K may be much less efficient than that for gp19K. Thus, the E3 genes may be organized such that the relative abundance of the 6.7K and gp19K proteins is controlled by the efficiency of their initiation codons in the same mRNA.

Adenoviruses, Human↗

PubMed1988

Determination of secondary structure in the initiation region of ovalbumin mRNA.

We have analyzed the secondary structure in the region surrounding the initiation codons of both cellular and synthetic versions of ovalbumin mRNA. RNase V1 cleavage sites and structure-dependent, chemically modified bases in cellular ovalbumin mRNA were determined by reverse transcription of hen poly A(+) RNA using ovalbumin-specific, synthetic DNA primers. These results indicate an extensive region of unpaired nucleotides preceding the initiation codon and a region of base-paired nucleotides including and following the initiation codon. A synthetic ovalbumin mRNA (SP65.OV) was prepared by run-off transcription of a cloned ovalbumin cDNA (pSP65.OV). Identical regions of hen ovalbumin and SP65.OV mRNAs gave identical patterns of structure-dependent base modifications. A computer program for determining RNA secondary structure was used to find a 5'-region structure for ovalbumin mRNA that is consistent with our data.

Animals↗

PubMed1998

Structure and organization of the Drosophila cholinergic locus.

The Drosophila cholinergic locus is composed of two distinct genetic functions: choline acetyltransferase (ChAT; EC 2.3.1.6), the enzyme catalyzing biosynthesis of neurotransmitter acetylcholine (ACh), and the vesicular ACh transporter (VAChT), the synaptic vesicle membrane protein which pumps transmitter into vesicles. Both genes share a common first exon and the remainder of the VAChT gene contains a single coding exon residing entirely within the first intron of ChAT. RNase protection analysis indicates that all Drosophila VAChT specific transcripts contain the shared first exon and suggests common transcriptional control for ChAT and VAChT. Similar types of genomic organization have been evolutionarily conserved for cholinergic loci in nematodes and vertebrates, and may operate to ensure coordinate expression of these functionally related genes in the same cells. The relative levels of Drosophila ChAT and VAChT mRNA differ, however, in different tissues or in Cha mutants, indicating that independent regulation of ChAT and VAChT transcripts may occur post-transcriptionally. The predicted Drosophila VAChT protein is composed of 578 amino acids and contains 12 conserved putative transmembrane domains. Full-length VAChT cDNA is 7.2 kilobase long and has unusually long 5'- and 3'-untranslated regions (UTR). The 5'-UTR contains a GTG ChAT translational initiation codon along with three other potential ATG initiation codons. These features of the VAChT 5'-UTR region suggest that a ribosome scanning model may not be used for VAChT translation initiation.

Amino Acid Sequence↗