Nucleotides that contribute to the identity of Escherichia coli tRNA(Phe)

A series of sequence variants of amber suppressor genes of tRNA(Phe) were synthesized in vitro and cloned in Escherichia coli to examine the contributions of individual nucleotides to identity for amino acid acceptance. Three different but complementary types of tRNA variants were constructed. The first involved the substitution of base-pairs on the cloverleaf stem regions of the E. coli tRNA(Phe). The second type of variant involved total gene synthesis based on wild-type tRNA(Phe) sequences found in Bacillus subtilis and in Halobacterium volcanii. In the third type of variant, the identity of E. coli tRNALys was changed to that of tRNA(Phe). The nucleotides which are important for tRNA(Phe) identity in E. coli are located on the corner of the L-shaped tRNA molecule, where the dihydrouridine loop interacts with the T loop, and extend to the interior opening of the anticodon stem and the adjoining variable loop. The nucleotide sequence on the dihydrouridine stem region, which joins the corner and stem regions, was not successfully studied though it may contribute to tRNA(Phe) identity. The fourth nucleotide from the 3' end of tRNA(Phe) has some importance for identity.     

Mutants affecting tRNA(Phe) from Escherichia coli. Studies of the suppression of thermosensitive phenylalanyl-tRNA synthetase

Four mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild-type pheV to complement the thermosensitive defect in a mutant of phenylalanyl-tRNA synthetase. One of these mutants, leading to the change C2----U2 in tRNA(Phe), is expressed about 10-fold lower in transformed cells than wild-type pheV. This mutant, unlike the remaining three (G15----A15, G44----A44, m7G46----A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number. The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity. A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl-tRNA synthetase. The mutant gene pheV (U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second-site mutation outside the tRNA structural gene, in the discriminator region. This mutation, C(-6)----T(-6), restores expression of the mutant U2 to about the level of wild-type tRNA(Phe).     

The pheR gene of Escherichia coli encodes tRNA(Phe), not a repressor protein

Nucleotide sequence analysis and transposon 5 (Tn5) insertional mutagenesis indicate that the Escherichia coli gene pheR encodes tRNA(Phe) and not a repressor protein as previously reported. The coding region of pheR is identical to that of three other cloned tRNA(Phe) genes, pheU, pheV, and pheW. Multicopy plasmids carrying pheR, like those carrying pheU, pheV, or pheW, complement a temperature-sensitive lesion in the gene for the alpha-subunit of phenylalanyl-tRNA synthetase (pheS). The nucleotide sequences of the 5'-flanking DNA of pheR, pheU, and pheW are almost identical but are quite different from the same region of pheV. By comparison with pheV, which has two tandem promoters, pheR was found to have a single promoter. The expression of pheA (encoding chorismate mutase/prephenate dehydratase) in strains carrying the pheR374 allele was decreased to similar extents by multicopy plasmids containing either pheR or pheV. It is proposed that this decrease in pheA expression and the increase in expression of pheA previously reported for chromosomal pheR mutants are both mediated through the attenuation control mechanism that regulates pheA.     

Dynamic equilibrium between the two conformational states of spin-labeled tropomyosin

Tropomyosin was labeled with a maleimide nitroxide spin-label attached to cysteine-190 via a succinimido ring which was subsequently opened by incubation at alkaline pH. Electron spin resonance (ESR) spectra showed a temperature-dependent equilibrium, below the main unfolding transition of tropomyosin, between labels which were restricted in their motion (strongly immobilized), predominating at low temperatures, and those which were highly mobile (weakly immobilized), predominating at higher temperatures. These label states were associated with two protein states from a comparison of the ESR spectral changes with the thermal unfolding profile of tropomyosin. The strongly immobilized labels were associated with the completely folded molded and the weakly immobilized labels with a partially unfolded (in the cysteine-190 region) state which is an intermediate in the thermal unfolding of tropomyosin. A spectral subtraction technique was used to measure the concentration ratio of strongly and weakly immobilized labels from which an equilibrium constant, K, was determined at different temperatures. A linear van't Hoff plot was obtained, indicating that the spin-labeled protein is in thermal equilibrium between these two conformational states with delta H = 17 kcal/mol, delta S = 56 cal/(deg X mol), and K = 1.0 at 34 degrees C. An upper limit of 10(7) s-1 for the conformational fluctuation was estimated from the shapes and separation of the two ESR spectral components. In contrast to the label with the opened succinimido ring, the spin-label with an intact succinimido ring remained strongly immobilized on the protein, indicating that in the partially unfolded state the molecule retains structure in the cysteine-190 region.     

Evidence that there are only two tRNA(Phe) genes in Escherichia coli.

pheV, one of the genes that code for tRNA(Phe), was deleted from the chromosome of a strain of Escherichia coli K-12. As a consequence of this mutation, expression of pheA, the gene for chorismate mutase P-prephenate dehydratase, the first enzyme in the terminal pathway of phenylalanine biosynthesis, was derepressed. Similar derepression of pheA has been reported in pheR mutants of E. coli K-12 (J. Gowrishankar and J. Pittard, J. Bacteriol. 150:1130-1137, 1982). Attempts to introduce a pheR mutation into the delta pheV strain failed under circumstances suggesting that this combination of mutations is lethal. Southern blot analysis of pheV+ and delta pheV strains indicated that there are only two tRNA(Phe) genes in E. coli. It is recommended that the names pheU and pheV be retained for these genes.     

tRNA binding sites of ribosomes from Escherichia coli

70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.     

Resolution of conformational states of spin-labeled myosin during steady-state ATP hydrolysis

We have measured the conventional electron paramagnetic resonance (EPR) spectrum of spin-labeled myosin filaments as a function of the nucleotide occupancy of the active site of the enzyme. The probe used was 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl (IASL), which reacts specifically with sulfhydryl 1 of the myosin head. In the absence of nucleotide, the probe remains strongly immobilized (rigidly attached to the myosin head) so that no nanosecond rotational motions are detectable. When MgADP is added to IASL-labeled myosin filaments (T = 20 degrees C), the probe mobility increases slightly. During steady-state MgADP hydrolysis (T = 20 degrees C), the probe undergoes large-amplitude nanosecond rotational motion. These results are consistent with previous studies of myosin monomers, heavy meromyosin, and myosin subfragment 1. Isoclinic points observed in overlays of sequential EPR spectra recorded during ATP hydrolysis strongly suggest that the probes fall into two motional classes, separated by approximately an order of magnitude in effective rotational correlation time. Both of the observed states are distinct from the conformation of myosin in the absence of nucleotides, and the spectrum of the less mobile population is indistinguishable from that observed in the presence of MgADP. The addition of ADP and vanadate to IASL-myosin gives rise to two motional classes virtually identical with those observed in the presence of ATP, but the relative concentrations of the spin populations are significantly different. We have quantitated the percentage of myosin in each motional state during ATP hydrolysis. The result agrees well with the predicted percentages in the two predominant chemical states in the myosin ATPase cycle. Spectra obtained in the presence of nucleotide analogues permit us to assign the conformational states to specific chemical states. We propose that the two motional classes represent two distinct local conformations of myosin that are in exchange with one another during the ATP hydrolysis reaction cycle.     

Poly(U)-dependent interaction of yeast tRNA(Phe) and its fragments with ribosomes from Escherichia coli

The poly(U)-dependent bindings of yeast tRNAPhe, its derivative depleted of 3'-terminal adenosine, and 15-nucleotide having a sequence of yeast tRNAPhe anticodon arm to the P site of Escherichia coli 70S ribosomes were compared. The equilibrium and rate constants were determined. Data indicate that the anticodon arm (N28-N42) contributes the major fraction of the binding free energy (-45.3 kJ/mol at 10 mM Mg2+ and 30 degrees C). Other parts of the tRNAPhe molecule besides A76 (N1-N27 and N43-N75) bring additional-6.0 kJ/mol, and A76 contributes-2.4 kJ/mol.     

Resolved conformational states of spin-labeled Ca-ATPase during the enzymatic cycle.

We have used frequency- and time-resolved electron paramagnetic resonance (EPR) to study the effects of substrate on the nanosecond conformational dynamics of the Ca-ATPase of sarcoplasmic reticulum, as detected by an iodoacetamide spin label (IASL) attached covalently to the enzyme. We confirm previous results [Coan, C. (1983) Biochemistry 22, 5826] showing that this probe is less rotationally mobile following the addition of nucleotides (ADP, AMPPNP, ATP) and that the shape of the spectrum suggests the presence of two components. We used two approaches to enhance EPR resolution in order to resolve the spectral components and their corresponding conformational states. First, to improve resolution in the frequency (spectral) domain, we used perdeuterated IASL, which results in narrower line widths. Digital spectral analysis resolves the EPR spectrum into two components, one that is indistinguishable from the spectrum observed in the absence of ligands and another that indicates more restricted probe motions, suggesting a distinct conformation of the labeled protein. Additions of substrate ligands appear to change only the mole fractions of the two components. The mole fraction of the restricted component (fR) was 0 in the absence of ligands, but increased to about 0.5 in the presence of saturating concentrations of AMPPNP and Ca2+. In general, ATP and its analogs increase fR, with larger effects observed in the presence of Ca. However, calcium has no effect by itself (fR = 0). Both monovanadate and decavanadate increase fR, but the formation of a covalent phosphoenzyme from inorganic phosphate (E2-P) had no effect (fR = 0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of a double missense mutation by a mutant tRNA(Phe) in Escherichia coli.

We report here the isolation of a mutant tRNAPhe that suppresses a double missense auxotrophic mutation in trpA of Escherichia coli, trpA218. The doubly mutant protein product differs from wild-type TrpA by the replacements of Phe22 by Leu and Gly211 by Ser. A partial revertant TrpA phenotype can be obtained from trpA218 by changing either Leu22 back to Phe or Ser211 back to Gly. Translational suppressors were previously obtained that act at codon 211, replacing the Ser211 in the TrpA218 protein, presumably with Gly. In the present study, we selected for trpA218 suppressors caused by mutation of a cloned tRNAPhe gene, pheV. DNA sequence analysis of the suppressor isolated reveals a singular structural alteration, changing the anticodon from 5'-GAA-3' to 5'-GAG-3'. Sequencing of trpA218 confirmed the likely identity of Leu22 as CUC. The new missense suppressor, designated pheV(SuCUC), is lethal to the cell when highly expressed, as from a high copy number plasmid. This may be due to efficient replacement of Leu by Phe at CUC (and, probably, CUU) codons throughout the genome. We anticipate that pheV(SuCUC) will prove, like other missense suppressors, to be extremely useful in studies on the specificity and accuracy of decoding.     

Kinetic mechanism of tRNA nucleotidyltransferase from Escherichia coli.

A kinetic analysis of the incorporation of AMP into tRNA lacking the 3'-terminal residue by tRNA nucleotidyltransferase (EC 2.2.7.25) from Escherichia coli is presented. Initial velocity studies demonstrate that the mechanism is sequential and that high concentrations of tRNA give rise to substrate inhibition which is noncompetitive with respect to ATP. In addition, the substrate inhibition is more pronounced in the presence of pyrophosphate, which suggests the formation of an inhibitory enzyme-pyrophosphate-tRNA complex. Noncompetitive product inhibition is observed between all possible pairs of substrates and products. ADP and alpha,beta-methylene adenosine triphosphate are competitive dead end inhibitors of ATP, while the latter is a noncompetitive dead end inhibitor of the tRNA substrate. A nonrapid equilibrium random mechanism is proposed which is consistent with these data and offers an explanation for the noncompetitive substrate inhibition by tRNA.