Differential polypeptide synthesis of the proton-translocating ATPase of Escherichia coli.

We investigated the regulation of the synthesis of the eight polypeptides of the Escherichia coli proton-translocating ATPase. A plasmid carrying the eight genes of the unc operon was used to direct in vivo and in vitro protein synthesis of the eight polypeptides. Analysis of these data indicates that the ATPase polypeptides are synthesized in unequal amounts both in vitro and in vivo. We identified several regions within the unc operon at which expression of a gene is either increased or decreased from that of the preceding gene. Since genetic information indicates a single polycistronic mRNA for all eight genes of this operon, the observed differential synthesis of the polypeptides is most likely the result of translational regulation. The effect of varying the temperature suggests that the secondary structure in the mRNA may affect the rate of translation initiation in the region between uncE and uncF.     

An acidic or basic amino acid at position 26 of the b subunit of Escherichia coli F1F0-ATPase impairs membrane proton permeability: suppression of the uncF469 nonsense mutation.

The uncF469 allele differed from normal in that a G----A base change occurred at nucleotide 77 of the uncF gene, resulting in a TAG stop codon rather than the tryptophan codon TGG. Two partial revertant strains were isolated which retained the uncF469 allele but formed a partially functional b-subunit, due to suppression of the uncF469 nonsense mutation. From the altered isoelectric points of the b-subunits from these strains, it was concluded that the suppressor gene of partial revertant strain AN1956 inserts an acidic amino acid for the TAG codon, and that the suppressor gene of partial revertant strain AN1958 inserts a basic amino acid. The membranes of both partial revertant strains showed impaired permeability to protons on removal of F1-ATPase. The membranes of both strains, however, were able to carry out oxidative phosphorylation, and the ATPase activities of both were resistant to the inhibitor dicyclohexylcarbodiimide.     

Effects of alterations in the translation control region on bacterial gene expression: use of cat gene constructs transcribed from the lac promoter as a model system

The region controlling translation of the cat gene, which codes for chloramphenicol acetyltransferase, has been varied structurally in a series of plasmids that place the gene under control of the lac promoter. These plasmid constructs have enabled study of the structural features that affect the efficiency of mRNA translation. Altering the potential for secondary structure formation within the translation control region caused a tenfold variation in the synthesis of CAT enzyme, whereas varying the distance between the Shine-Dalgarno sequence (SD) and the translation start codon from 7 to 13 bases did not significantly affect the yield of CAT. If the SD was situated in a region of mRNA that is capable of base pairing, the efficiency of translation was decreased; however, the translation start codon, AUG, can initiate translation efficiently even when located in a segment capable of duplex formation. Overlapping of the cat translation control region by translation initiated upstream markedly affected initiation of translation within the cat gene: out-of-frame overlapping translation reduced CAT production by 90%; in-frame overlapping translation prevented detectable initiation of protein synthesis at the cat gene translation start codon, and yielded only fusion proteins. The enzymatic activity of such proteins was influenced by the length of the adventitious peptide segment added to the amino-terminus of the CAT polypeptide.     

Complementation between uncF alleles affecting assembly of the F1F0-ATPase complex of Escherichia coli.

A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo. The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome. The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation. Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable. It was concluded that the F0 sector was assembled. Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131. Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp). Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages.     

Targeted mutagenesis of the b subunit of F1F0 ATP synthase in Escherichia coli: Glu-77 through Gln-85.

Subunit b of Escherichia coli F1F0 ATP synthase contains a large hydrophilic region thought to be involved in the interaction between F1 and F0. Oligonucleotide-directed mutagenesis was used to evaluate the functional importance of a segment of this region from Glu-77 through Gln-85. The mutagenesis procedure employed a phagemid DNA template and a doped oligonucleotide primer designed to generate a predetermined collection of missense mutations in the target segment. Sixty-one mutant phagemids were identified and shown to contain nucleotide substitutions encoding 37 novel missense mutations. Mutations were isolated singly or in combinations of up to four mutations per recombinant phagemid. F1F0 ATP synthase function was studied by mutant phagemid complementation of a novel E. coli strain in which the uncF (b) gene was deleted. Complementation was assessed by observing growth on solid succinate minimal medium. Many phagemid-encoded uncF (b) gene mutations in the targeted segment resulted in growth phenotypes indistinguishable from those of strains expressing the native b subunit, suggesting abundant F1F0 ATP synthase activity. In contrast, several specific mutations were associated with a loss of enzyme function. Phagemids specifying the Ala-79----Pro, Arg-82----Pro, Arg-83----Pro, or Gln-85----Pro mutation failed to complement uncF (b) gene-deficient E. coli. F1F0 ATP synthase displayed the greatest sensitivity to mutations altering a single site in the target segment, Ala-79. The evidence suggests that Ala-79 occupies a restricted position in the enzyme complex.     

Ribosomal binding to the internal ribosomal entry site of classical swine fever virus

Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is approximately 330 nt long, highly structured, and mediates internal initiation of translation with no requirement for elF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363-391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.     

The study of spermidine-stimulated polypeptide synthesis in cell-free translation of mRNA from lactating mouse mammary gland

The stimulatory effect of spermidine on the translation of poly(A)⁺ mRNA from lactating mouse mammary glands in a wheat germ system was studied. Spermidine stimulated total polypeptide synthesis about 2.5-fold relative to that occurring in the presence of an optimal concentration of Mg²⁺ alone. The size and the number of polysomes were about 1.6-times larger in the presence of spermidine than in its absence. A similar magnitude of increase in peptide chain initiation, 1.4-fold, was found when the extent of peptide chain initiation was measured by determining the residual polypeptide synthesis subsequent to the addition of inhibitor(s) of peptide chain initiation to the in vitro translation system with or without spermidine at various times of the incubation. Time-course study of the release of polypeptide from polysomes showed that spermidine stimulated this process to a much greater extent than peptide chain initiation, indicating that the polyamine also increases the rate of peptide chain elongation. The extent of stimulation of peptide chain elongation by spermidine was estimated to be about 1.5-fold when the disappearance of isotope-labeled nascent peptides from polysomes was measured by pulse-chase experiments. These results indicate that spermidine stimulates the cell-free translation of mammary mRNA by increasing the rates of both initiation and elongation of polypeptide synthesis to almost the same extent. The polyamine also reduced the relative amount of incomplete polypeptides, thereby increasing the yield of full-length translational products.     

UGA nonsense mutation in the alcohol dehydrogenase gene of Drosophila melanogaster

A mutant gene, which we have designated AdhnB, codes for a defective form of the enzyme alcohol dehydrogenase in Drosophila melanogaster. We show that the polypeptide encoded by AdhnB is approximately 2000 Mr smaller than the protein synthesized under the direction of the wild-type alcohol dehydrogenase gene. In contrast, the alcohol dehydrogenase mRNA produced by both genes is the same size. We cloned and sequenced a portion of the protein-coding region of AdhnB and compared it to the same region in the wild-type gene. We found a single base substitution: a change of the TGG tryptophan codon at amino acid 235 to a TGA termination codon. This nonsense mutation accounts for the observed reduction in size of the alcohol dehydrogenase polypeptide. In further studies, we found that the steady-state levels of alcohol dehydrogenase mRNA in flies carrying the AdhnB gene and the wild-type alcohol dehydrogenase gene were indistinguishable. However, the steady-state level of alcohol dehydrogenase polypeptide was reduced to 1% of wild-type levels in flies with the AdhnB gene. Moreover, the rate of alcohol dehydrogenase synthesis in mutant flies was reduced to 50% of that found in wild type. The aberration in AdhnB thus affects both the rate of synthesis and the rate of degradation of the alcohol dehydrogenase peptide. AdhnB is the first reported nonsense mutant in Drosophila.     

An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function.

Glycine at position 9 is replaced by aspartic acid in the mutant b-subunit of Escherichia coli F1F0-ATPase coded for by the uncF476 allele. The mutant b-subunit is not assembled into the membrane in haploid strains carrying the uncF476 allele, but, if the mutant allele is incorporated into a multicopy plasmid, then some assembly of the mutant b-subunit occurs. Two revertant strains were characterized, one of which (AN2030) was a full revertant, the other (AN1953) a partial revertant. DNA sequencing indicated that in strain AN2030 the uncF476 mutation had reverted to give the sequence found in the normal uncF gene. The partial-revertant strain AN1953, however, retained the DNA sequence of the uncF476 allele, and complementation analysis indicated that the second mutation may be in the uncA gene. Membranes prepared from the partial-revertant strain carried out oxidative phosphorylation, although the membranes appeared to be impermeable to protons, and the ATPase activity was sensitive to the inhibitor dicyclohexylcarbodi-imide.     

Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide.

Previous nucleotide sequence analysis of RNA segment 7 of influenza B virus indicated that, in addition to the reading frame encoding the 248 amino acid M1 protein, there is a second overlapping reading frame (BM2ORF) of 585 nucleotides that has the coding capacity for 195 amino acids. To search for a polypeptide product derived from BM2ORF, a genetically engineered beta-galactosidase-BM2ORF fusion protein was expressed in Escherichia coli and a polyclonal rabbit antiserum was raised to the purified fusion protein. This antiserum was used to identify a polypeptide, designated BM2 protein (Mr approximately equal to 12,000), that is synthesized in influenza B virus-infected cells. To understand the mechanism by which the BM2 protein is generated from influenza B virus RNA segment 7, a mutational analysis of the cloned DNA was performed and the altered DNAs were expressed in eukaryotic cells. The expression patterns of the M1 and BM2 proteins from the altered DNAs indicate that the BM2 protein initiation codon overlaps with the termination codon of the M1 protein in an overlapping translational stop-start pentanucleotide, TAATG, and that the expression of the BM2 protein requires 5'-adjacent termination of M1 synthesis. Our data suggest that a termination-reinitiation scheme is used in translation of a bicistronic mRNA derived from influenza B virus RNA segment 7, and this strategy has some analogy to prokaryotic coupled stop-start translation of tandem cistrons.     

Stimulation of the protein synthetic process by adenosine 3':5'-monophosphate and hexose phosphates in gel-filtered rabbit reticulocyte lysates.

The addition of 0.167 to 4.0 mM cAMP to gel-filtered rabbit reticulocyte lysates stimulates the initial rate and the extent of polypeptide synthesis. The stimulation is at the initiation step of polypeptide synthesis as measured by the (i) increased dipeptide, methionyl-valine, accumulation in the presence of the specific initiation inhibitor, pactamycin, and (ii) increased formation of the 40 S and 80 S initiation complex when gel-filtered lysates are incubated with [35S]Met-tRNAFMet. Furthermore, a synergistic stimulation of protein synthesis is observed when cAMP and hexose phosphates (which alone elicit a 1.8-fold stimulation of protein synthesis) are added simultaneously to gel-filtered rabbit reticulocyte lysates. These results indicate that cAMP and hexose phosphates are both essential to maintain the high rate of initiation.     

High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter

Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter (pL) of bacteriophage lambda. Upon temperature induction the best of our constructions expressed a small-t-related 19 000-dalton polypeptide in an amount corresponding to approx. 2.5% of total de novo protein synthesis. This 19 000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis. In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent Mr of 14 500. This 14 500-dalton polypeptide was shown to be related to authentic small-t. Presumably the secondary structure of the mRNA starting at pL is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon.     

Effect of mutations and deletions in a bicistronic mRNA on the synthesis of influenza B virus NB and NA glycoproteins.

The mRNA derived from influenza B virus RNA segment 6 is functionally bicistronic and encodes the NB and NA glycoproteins in different, overlapping reading frames. NB protein synthesis is initiated at the 5'-proximal AUG codon, and 4 nucleotides downstream there is a second AUG codon which is used to initiate NA protein synthesis. The nucleotide sequence context of the first AUG codon conforms closely with the established 5'-CC(A/G)CCAUGG-3' consensus sequence (M. Kozak, Nucleic Acids Res. 15:8125-8148, 1987), which should favor initiation of NB protein synthesis at this site, yet NB and NA are found to accumulate in approximately equal amounts in infected cells. To determine the features important for allowing initiation at the second 5'-proximal AUG codon, we made changes in the 5'-terminal region of the mRNA, including deletions, insertions, and site-specific mutations. The recombinant DNA molecules were expressed in eucaryotic cells, and the accumulation of NB and NA was quantitated. The data indicate that changes in the immediate sequence around the first AUG codon do not make a large difference in the amounts of NB and NA that accumulate, but that when the first AUG codon is displaced from its normal position it is now quite efficient at preventing downstream initiation events. In addition, the data indicate that an element of the B/NB/NA mRNA 5' untranslated leader region acts in cis to enhance the expression of NB and NA.     

GUG is an efficient initiation codon to translate the human mitochondrial ATP6 gene

A maternally inherited and practically homoplasmic mitochondrial (mtDNA) mutation, 8527A>G, changing the initiation codon AUG into GUG, normally coding for a valine, was observed in the ATP6 gene encoding the ATPase subunit a. No alternate Met codon could replace the normal translational initiator. The patient harboring this mutation exhibited clinical symptoms suggesting a mitochondrial disease but his mother who carried the same mtDNA mutation was healthy. The mutation was absent from 100 controls and occurred once amongst 44 patients suspected of Leber Hereditary Optic Neuropathy (LHON) but devoid of typical LHON mutations. In patient fibroblasts, no effect of 8527A>G mutation could be demonstrated on the biosynthesis of mtDNA-encoded proteins, on size and the content of ATPase subunit a, on ATP hydrolysis and on mitochondrial membrane potential. In addition, ATP synthesis was barely decreased. Therefore, GUG is a functional initiation codon for the human ATP6 gene.     

Multiple glycoproteins synthesized by the smallest RNA segment (S10) of bluetongue virus.

The genome of bluetongue virus, an orbivirus, consists of 10 double-stranded RNAs, each encoding at least one polypeptide. The smallest RNA segment (S10) encodes two minor nonstructural proteins, NS3 and NS3A, the structures and functions of which are not understood. We have expressed these two proteins in mammalian cells by using the T7 cytoplasmic transient expression system. Using a deletion mutant (lacking the first AUG initiation codon), we have demonstrated that the second initiation codon is used to initiate the synthesis of NS3A protein and that the two initiation codons are responsible for the synthesis not only of NS3 and NS3A but also of high-molecular-weight forms of both proteins. These higher-molecular-weight forms (GNS3 and GNS3A) are glycosylated. We have also demonstrated that the carbohydrate chains of GNS3 and GNS3A could be further modified by heterogeneous extension to polylactosaminoglycan forms. The glycosylated and nonglycosylated forms are found in similar intracellular locations in the Golgi complex. In the presence of cycloheximide, NS3 and NS3A immunofluorescence staining was pronounced in the Golgi complex, confirming that NS3 and NS3A are competent for transport to the Golgi apparatus after synthesis. We conclude that S10 gene products are integral membrane glycoproteins.     

The influence of mRNA primary and secondary structure on human IFN-gamma gene expression in E. coli.

Parameters influencing the efficiency of expression of the human immune interferon (IFN-gamma) gene in E. coli were studied by comparing a series of eight in vitro-derived gene variants. These contained all possible combinations of silent mutations in the first three codons of the mature IFN-gamma polypeptide coding sequence. Expression levels varied up to 50-fold among the different constructions. Comparison of messenger RNA secondary structure models for each variant suggested that the presence of stem-loop structures blocking the translation initiation signals could drastically decrease the efficiency of IFN-gamma synthesis. With variants displaying no stable mRNA secondary structure in the region, a C----U transition at position +11 after the AUG resulted in a 5-fold increase in expression indicating that RNA primary structure also plays an important role in expression. In addition we demonstrate that, in this system, a spacing of 8 nucleotides between the Shine-Dalgarno region and AUG was optimal for gene expression and that the steady-state production level of IFN-gamma rose exponentially with increasing rate of synthesis.