An upstream uncD sequence modulates translation of Escherichia coli uncC.

The effect of upstream uncD sequences on expression of the Escherichia coli uncC gene, encoding the epsilon subunit of F1-ATPase, was studied. uncC expression was reduced severalfold in plasmid constructs bearing, in addition to uncC, a region of uncD located between 85 and 119 bases upstream from the uncC initiation codon. This reduction was independent of in-frame translation of the uncD sequences. An mRNA stem-loop structure in which sequences located within the inhibitory region of uncD base pair with the uncDC intercistronic region is suggested to function in modulating uncC expression.     

Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits.

A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed.     

Altered translation of the uncC gene coding for the epsilon subunit of the F1F0-ATPase of Escherichia coli.

The nucleotide sequence of the previously described uncC424 allele was determined and found to be the same as that of a wild-type uncC gene. However, a G----A change occurred 7 nucleotides upstream from the translation start codon, changing the putative Shine-Dalgarno sequence from GAGG to GAAG. Four revertant strains were examined. In one revertant, which had normal growth and membrane properties, a single base deletion had occurred to re-form the Shine-Dalgarno sequence GAGG 1 nucleotide closer to the translation start codon. A second revertant had a single base deletion in the preceding uncD gene, causing an extension of the beta subunit by 6 amino acids and an increase, presumably by translational coupling, in the amount of epsilon subunit. The third and fourth revertant strains were phenotypically similar and had either C----T or G----T changes 18 or 19 nucleotides, respectively, upstream from the translation start codon.     

Assembly of a functional F1 of the proton-translocating ATPase of Escherichia coli

Assembly of the F1 portion of the proton-translocating ATPase of Escherichia coli was examined in vivo. Analysis of strains lacking genes which specify the Fo polypeptides a, b, and c showed that the F1 subunits were able to assemble into a complex in the absence of the Fo subunits. In addition we have investigated the effects of mutations in the individual genes which specify the F1 polypeptides on the assembly process. Mutations of the uncA(alpha), uncG(gamma), or uncD(beta) genes result in a defective assembly of the F1 complex. In contrast, mutations in the uncH(delta) or uncC(epsilon) genes did not prevent assembly of the core alpha beta gamma complex. In these cases, however, the partial F1 complexes were incapable of restoring energy-linked functions to F1-depleted membranes.     

Role of the amino terminal region of the epsilon subunit of Escherichia coli H(+)-ATPase (F0F1).

Escherichia coli strain KF148(SD-) defective in translation of the uncC gene for the epsilon subunit of H(+)-ATPase could not support growth by oxidative phosphorylation due to lack of F1 binding to Fo (M. Kuki, T. Noumi, M. Maeda, A. Amemura, and M. Futai, 1988, J. Biol. Chem. 263, 17, 437-17, 442). Mutant uncC genes for epsilon subunits lacking different lengths from the amino terminus were constructed and introduced into strain KF148(SD-). F1 with an epsilon subunit lacking the 15 amino-terminal residues could bind to F0 in a functionally competent manner, indicating that these amino acid residues are not absolutely necessary for formation of a functional enzyme. However, mutant F1 in which the epsilon subunit lacked 16 amino-terminal residues showed defective coupling between ATP hydrolysis (synthesis) and H(+)-translocation, although the mutant F1 showed partial binding to Fo. These findings suggest that the epsilon subunit is essential for binding of F1 to F0 and for normal H(+)-translocation. Previously, Kuki et al. (cited above) reported that 60 residues were not necessary for a functional enzyme. However, the mutant with an epsilon subunit lacking 15 residues from the amino terminus and 4 residues from the carboxyl terminus was defective in oxidative phosphorylation, suggesting that both terminal regions affect the conformation of the region essential for a functional enzyme.     

In vivo evidence for the role of the epsilon subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli

The function of the epsilon subunit of the Escherichia coli proton-translocating ATPase has been examined by using a mutant defective in the uncC gene. Strains with a defective uncC gene show a reduction in both growth yield and growth rate that is more severe than for other unc mutants; this deleterious effect is shown to be a result of the ATPase activity of the F1 complex which is missing the epsilon subunit. In addition, the epsilon-deficient F1 is bound less tightly to the membrane. These data suggest that, in vivo, the epsilon subunit is capable of inhibiting the ATPase activity of F1 and also functions in the binding of F1 to F0.     

Cost minimization of ribosomal frameshifts

Properties of mRNA leading regions that modulate protein synthesis are little known (besides effects of their secondary structure). Here I explore how coding properties of leading regions may account for their disparate efficiencies. Trinucleotides that form off frame stop codons decrease costs of ribosomal slippages during protein synthesis: protein activity (as a proxy of gene expression, and as measured in experiments using artificial variants of 5' leading sequences of beta galactosidase in Escherichia coli) increases proportionally to the number of stop motifs in any frame in the 5' leading region. This suggests that stop codons in the 5' leading region, upstream of the recognized coding sequence, terminate eventual translations that sometimes start before ribosomes reach the mRNA's recognized start codon, increasing efficiency. This hypothesis is confirmed by further analyses: mRNAs with 5' leading regions containing in the same frame a start preceding a stop codon (in any frame) produce less enzymatic activity than those with the stop preceding the start. Hence coding properties, in addition to other properties, such as the secondary structure of the 5' leading region, regulate translation. This experimentally (a) confirms that within coding regions, off frame stops increase protein synthesis efficiency by early stopping frameshifted translation; (b) suggests that this occurs for all frames also in 5' leading regions and that (c) several alternative start codons that function at different probabilities should routinely be considered for all genes in the region of the recognized initiation codon. An unknown number of short peptides might be translated from coding and non-coding regions of RNAs.     

Stability of structures of the epsilon subunit and terminator of thermophilic ATPase

F1-type ATPase is the central enzyme for ATP synthesis in most organisms. Because of the extreme reconstitutability of thermophilic ATPase (TF1) and diversity of the minor subunits of F1 type ATPase, an operon coding for TF1 was isolated from DNA of thermophilic bacterium PS3, and its terminal region containing the epsilon subunit (TF1 epsilon) and terminator was sequenced. The primary structure of the epsilon subunit (Mr = 14 333) was deduced from the nucleotide sequence (396 base-pairs) and amino-acid sequence of its amino terminus. The conclusions drawn from the results are as follows. Homologies: TF1 epsilon shows only 6% homology with the epsilon subunits of eight species reported, but 50% homology with Escherichia coli epsilon and 41% with chloroplast. The residues having a tendency to form reverse turns (Gly, Pro and Tyr) and His are relatively well conserved. Unlike some F1 epsilon types TF1 epsilon has no ATPase inhibitor activity and is not homologous with ATPase inhibitor. TF1 epsilon is essential to connect F1 to F0, like the b subunit, and is weakly homologous with the b subunit of F0F1. The cause of 3 beta: 1 epsilon subunit stoichiometry: The ribosome binding sequence of TF1 epsilon is TAGGN7, which is incomplete compared with that of TF1 beta. The codon usage for TF1 epsilon is similar to that for TF1 epsilon. The cause of stability of TF1 epsilon and its gene: There are 18 ionic groups at the putative reverse turns and the N- and C-termini of TF1 epsilon, but only 10 ionic groups in the corresponding sites of E. coli epsilon subunit. These ionic groups enhance the external polarity of TF1 epsilon and may intensify subunit-subunit interaction. There is a terminator at the 3' end of the TF1 epsilon gene, which is stabilized by a long (13 base-pairs) stem.     

Construction and plasmid-borne complementation of strains lacking the epsilon subunit of the Escherichia coli F1F0 ATP synthase.

Two strains of Escherichia coli that lack the epsilon subunit of the F1F0 ATP synthase have been constructed. They are shown to be viable but with very low growth yields (28%). These strains can be complemented by plasmids carrying wild-type uncC, but not when epsilon is overproduced. These results indicate that epsilon is not essential for growth on minimal glucose medium and that the level of its expression affects the assembly of the ATP synthase.     

Stable structure of thermophilic proton ATPase beta subunit

F1-ATPase is the major enzyme for ATP synthesis in mitochondria, chloroplasts, and bacterial plasma membranes. F1-ATPase obtained from thermophilic bacterium PS3 (TF1) is the only ATPase which can be reconstituted from its primary structure. Its beta subunit constitutes the catalytic site, and is capable of forming hybrid F1's with E. coli alpha and gamma subunits. Since the stability of TF1 resides in its primary structure, we cloned a gene coding for TF1, and the primary structure of the beta subunit was deduced from the nucleotide sequence of the gene to compare the sequence with those of beta's of three major categories of F1's; prokaryotic membranes, chloroplasts, and mitochondria. The following results were obtained. Homology: The primary structure of the TF1 beta subunit (473 residues, Mr = 51,995.6) showed 89.3% homology with 270 residues which are identical in the beta subunits from human mitochondria, spinach chloroplasts, and E. coli. It contained regions homologous to several nucleotide-binding proteins. Secondary structure: The deduced alpha-helical (30.1%) and beta-sheet (22.3%) contents were consistent with those determined from the circular dichroism spectra. Residues forming reverse turns (Gly and Pro) were highly conserved among the F1 beta subunits. Substituted residues and stability of TF1: We compared the amino acid sequence of the TF1 beta subunit with those of the other F1 beta subunits mentioned above. The observed substitutions in the thermophilic subunit increased its propensities to form secondary structures, and its external polarity to form tertiary structure. Codon usage: The codon usage of the TF1 beta gene was found to be unique. The changes in codons that achieved these amino acid substitutions were much larger than those caused by minimal mutations, and the third letters of the optimal codons were either guanine or cytosine, except in codons for Gln, Lys, and Glu.     

Catalytic control and coupling efficiency of the Escherichia coli FoF1 ATP synthase: influence of the Fo sector and epsilon subunit on the catalytic transition state

The rate-limiting transition state of steady-state ATP hydrolysis and synthesis reactions in the F(o)F(1) ATP synthase involves the rotation of the gamma, epsilon, and c subunits. To probe the role of the transport and coupling mechanisms in controlling catalysis, kinetic and thermodynamic parameters of ATP hydrolysis were determined for enzymes in the presence of the detergent lauryldimethylamine oxide (LDAO), which uncouples active transport and disables the inhibitory effect of the epsilon subunit. At 5 mM LDAO or greater, the inhibitory effects of epsilon subunit are abrogated in both purified F(1) and membranous F(o)F(1). In these conditions, LDAO solubilized F(o)F(1) has a higher k(cat) for ATP hydrolysis than F(1). These results indicate an influence of F(o) on F(1) even though catalysis is uncoupled from transport. The alpha(3)beta(3)gamma complex free of the epsilon subunit is activated at a lower concentration of 0.5 mM LDAO. Significantly, the gammaY205C mutant enzyme is similarly activated at 0.5 mM LDAO, suggesting that the mutant enzyme lacks epsilon inhibition. The gammaY205C F(o)F(1), which has a k(cat) for ATP hydrolysis 2-fold higher than wild type, has an ATP synthesis rate 3-fold lower than wild type, showing that coupling is inefficient. Arrhenius and isokinetic analyses indicate that enzymes that are free of epsilon subunit inhibition have a different transition-state structure from those under the influence of the epsilon subunit. We propose that the epsilon subunit is one of the factors that determines the proper transition-state structure, which is essential for efficient coupling.     

Overlap and cotranscription of the genes for the beta and epsilon subunits of tobacco chloroplast ATPase

The nucleotide sequences of the genes for the beta and epsilon subunits of tobacco chloroplast ATPase have been determined. The coding regions for the beta and epsilon subunits contain 1494 bp (498 codons) and 399 bp (133 codons), respectively. The 3' end of the beta-coding region overlaps by one nucleotide with the 5' end of the epsilon-coding region. The overlapping termination and initiation codons are ATGA. The beta and epsilon genes are cotranscribed as a 2.7-kb polycistronic mRNA. The amount of the beta and epsilon mRNA in the chloroplast is about one-twentieth that of the LS mRNA.     

Conformational interactions between alpha and beta subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes

In contrast to wild-type F1 adenosine triphosphatase, the beta subunits of soluble ATPase from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol-specific reagents 5-iodoacetamidofluorescein, 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid or 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-diazole. The mutation in the alpha subunit (uncA401) of F1 ATPase thus influences the accessibility of the single cysteinyl residue in the beta subunit. Following reaction of ATPase with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or N,N'-dicyclohexylcarbodiimide, the alpha and beta subunits of the uncA401, but not of the uncD412 mutant F1 ATPase were intensely labeled by a fluorescent thiol reagent. The mutation in the beta subunit (uncD412) thus influences the accessibility of the cysteinyl residues in the alpha subunit. In other work [Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 248] we have shown that 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid react with a different beta subunit from that labeled by N,N'-dicyclohexylcarbodiimide. This asymmetry with respect to modification by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N'-dicyclohexylcarbodiimide was seen in both mutant enzymes. In addition, the modification of one beta subunit of the uncA401 F1 ATPase induced the previously unreactive sulfhydryl group of another beta subunit to react with 2-(4'-iodoacetamidoanilino-naphthalene-6-sulfonic acid. These results provide evidence for at least three types of conformational interactions of the major subunits of F1 ATPase: from alpha to beta, from beta to alpha, and from beta to beta. As in wild-type ATPase, labeling of membrane-bound unc mutant ATPase by a fluorescent thiol reagent modified the alpha subunits. This suggests that a conformational change of yet a different type occurs when the enzyme binds to the membrane.     

Characterization of semi-uncoupled hybrid Escherichia coli-Bacillus megaterium F1F0 proton-translocating ATPases.

Cloned atp genes for the proton-translocating ATPase of the obligate aerobe Bacillus megaterium have been demonstrated to be capable of complementing Escherichia coli ATPase (unc) mutants (Hawthorne, C. A., and Brusilow, W. S. A. (1986) J. Biol. Chem. 261, 5245-5248). To determine the minimum subunit requirements for cross-species complementation, we constructed all combinations of B. megaterium atpA, G, D, and C genes (coding for the alpha, gamma, beta, and epsilon subunits, respectively) and tested their abilities to complement two uncA (alpha subunit) and two uncD (beta subunit) mutants of E. coli. The results indicated that complementation of either uncD mutant required atpD (beta) only. Complementation of one of the uncA (alpha) mutants required atpA, G, and D (alpha, gamma, and beta) and possibly atpE (epsilon) as well. The other uncA mutant was not complemented by any combination of B. megaterium ATPase genes. Complementation of a beta mutant by atpD (beta) or atpD and C (beta epsilon) produced cells which could grow aerobically on a nonfermentable carbon source (succinate) but not anaerobically on rich medium containing glucose. These E. coli therefore had become obligate aerobes. The ability to grow anaerobically could be restored to the mutant complemented by atpD alone by growth at pH 7.5 or pH 8 in the presence of 0.1 M potassium.     

Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein

Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression. Termination-reinitiation is dependent upon 45 nucleotide (nt) of RNA immediately upstream of the UAAUG pentanucleotide, which includes an essential stretch complementary to 18S rRNA helix 26. Thus, similar to the caliciviruses, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the M1 stop codon. Consistent with this, repositioning of the M1 stop codon more than 24 nt downstream from the BM2 start codon inhibited BM2 expression. RNA structure probing revealed that the RNA upstream of the UAAUG overlap is not highly structured, but upon encountering the M1 stop codon by the ribosome, a stem-loop may form immediately 5' of the ribosome, with the 18S rRNA complementary region in the apical loop and in close proximity to helix 26. Mutational analysis reveals that the normal requirements for start site selection in BM2 expression are suspended, with little effect of initiation codon context and efficient use of noncanonical initiation codons. This suggests that the full complement of initiation factors is not required for the reinitiation process.     

Chemomechanical Coupling in Single-Molecule F-Type ATP Synthase

An extremely small reaction chamber with a volume of a few femtoliters was developed for a highly sensitive detection of biological reaction. By encapsulating a single F(1)-ATPase (F(1)) molecule with ADP and an inorganic phosphate in the chamber, the chemomechanical coupling efficiency of ATP synthesis catalyzed by reversely rotated F(1) was successfully determined (Rondelez et al., 2005a, Nature, 444, 773-777). While the alpha3beta3gamma subcomplex of F(1) generated ATP with a low efficiency (approximately 10%), inclusion of the epsilon subunit into the subcomplex enhanced the efficiency up to 77%. This raises a new question about the mechanism of F(0)F(1)-ATP synthase (F(0)F(1)): How does the epsilon subunit support the highly coupled ATP synthesis of F(1)? To address this question, we measured the conformational dynamics of the epsilon subunit using fluorescence resonance energy transfer (FRET) at the single-molecule level. The experimental data revealed epsilon changes the conformation of its C-terminus helices in a nucleotide-dependent manner. It is plausible that the conformational change of epsilon switches the catalytic mode of F(0)F(1) for highly coupled ATP synthesis.