Nuclear magnetic resonance of E. coli ribosomes and viruses.

¹H Nuclear Magnetic Resonance spectra of a number of viruses and E. Coli ribosomes revealed that experimental values of the linewidth (πT₂)^?1 (< 320 Hz) and T₁ (< 1 sec) of the observable nuclei are too small to be accounted for by the system's molecular weight. The nuclei therefore must be internally mobile. From ¹³C Nuclear Magnetic Resonance spectra of 12% ¹³C enriched E. Coli ribosomes, it follows that 30% of the 5000 $\text{CH}{}_2\text{CH}{}_3$ groups, 10–20% of the 225 Phe residues, 20% of the δ-Arg and β-Lys carbons, ～ 100 nucleotides and a number of C_α carbons are internally mobile. It is demonstrated that ¹³C Nuclear Magnetic Resonance can fruitfully be applied to intact ribosomes.     

Nuclear magnetic resonance studies of formyltetrahydrofolate synthetase interactions with formate and methylammonium ion

Using nuclear magnetic resonance techniques, we have measured the internuclear distances separating the nucleotide-bound metal from the carbon and hydrogen nuclei of formate as well as the carbon of methylammonium cation when bound to formyltetrahydrofolate synthetase. Measurements were made of the paramagnetic effect on the spin-lattice relaxation rates (1/T1) of 13C and 1H nuclei arising from the replacement of Mg2+ with Mn2+, which binds to the enzyme in the form of a metal-nucleotide complex. Distances from Mn2+ to the formate carbon and proton were found to be 6.3 and 7.4 A, respectively, in the E . ATP . Mn2+ . formate complex and 6.0 and 7.1 A, respectively, in the E . ADP . Mn2+ . formate complex. When tetrahydrofolate was added to the latter complex, the exchange of formate was greatly reduced and became rate limiting for relaxation. These results are consistent with substantial conformational effects produced by the binding of the cofactor. The distance from Mn2+ to the methylammonium carbon in the E . ADP . Mn2+ . CH3NH+3, E . ADP . Mn2+ . formate . CH3NH3+, and E . ADP . Mn2+ . tetrahydrofolate . CH3NH3+ complexes was estimated to be in the range of 7.4-12 A. However, in the E . ADP . Mn2+ formate . tetrahydrofolate . CH3NH3+ complex, the data suggest that exchange of cation contributes significantly to relaxation. These results, combined with other known features of the enzyme, suggest that there may be a monovalent cation site within the active site of the enzyme.     

Glutamylsulfamoyladenosine and pyroglutamylsulfamoyladenosine are competitive inhibitors of E. coli glutamyl-tRNA synthetase

5'-O-[N-(L-glutamyl)-sulfamoyl] adenosine is a potent competitive inhibitor of E. coli glutamyl-tRNA synthetase with respect to glutamic acid (K(i) = 2.8 nM) and is the best inhibitor of this enzyme. It is a weaker inhibitor of mammalian glutamyl-tRNA synthetase (K(i) = 70 nM). The corresponding 5'-O-[N-(L-pyroglutamyl)-sulfamoyl] adenosine is a weak inhibitor (K(i) = 15 microM) of the E. coli enzyme.     

Monomeric structure of glutamyl-tRNA synthetase in Escherichia coli.

An investigation of the subunit structure of glutamyl-tRNA synthetase (EC 6.1.1.17) from Escherichia coli indicates that this enzyme is a monomer. The enzyme purified to apparent homogeneity is a single polypeptide chain with a molecular weight of 62,000 ± 3,000 and K^Glu_m ? 50 μM in the aminoacylation reaction. Analytical gel electrophoretic procedures were used to determine the molecular weight of species exhibiting glutamyl-tRNA synthetase activity in freshly prepared extracts of several strains of E. coli, which had been grown under various nutritional conditions and harvested at different stages of growth. In all cases, glutamyl-tRNA synthetase activity was associated with a protein having about the same molecular weight and K^Glu_m as the purified enzyme. Thus, no evidence of an oligomeric form of glutamyl-tRNA synthetase with a greater affinity for l-glutamate was obtained, in contrast to a previous report of J. Lapointe and D. Söll (J. Biol. Chem.247, 4966–4974, 1972).     

The glutamyl-tRNA synthetase of Escherichia coli: substrate-induced protection against its thermal inactivation.

The substrates-induced protection against the heat-inactivation of the glutamyl-tRNA synthetase has been investigated. tRNAGlu and ATP protect efficiently the enzyme, whereas glutamate does not. In the presence of tRNAGlu, glutamate induces an additional protection to that given by the tRNAGlu alone. A weak synergism was observed between ATP and tRNAGlu, whereas no synergism was detected between ATP and glutamate. These results suggest that tRNAGlu and ATP, but not glutamate are able to bind to the free enzyme form; glutamate binds only to the Enzyme.tRNAGlu and to the Enzyme.tRNAGlu.ATP complexes. The presence of the three substrates induces a higher stabilization of the enzyme than that expected from the protection observed for the various other substrates combinations, suggesting the existence of a marked synergism between the three substrates against the heat-inactivation of the enzyme. The protection constants determined from this study are similar to the dissociation constants determined by direct binding experiments and to the Km values determined kinetically.     

Sequence of E. coli tRNA-Glu1 by automated sequential degradation.

A minor tRNA-Glu1 constituent of a preparation of E. coli B tRNA-Glu (Oak Ridge) has a guanosine residue at position 66 rather than an adenosine as in the tRNA-Glu2 described by Ohashi et al. (3). Automated sequential degradation was used to sequence this region.     

Methylphosphonates as probes of protein-nucleic acid interactions.

Deoxydinucleoside methylphosphonates were prepared by chemical synthesis and were introduced stereospecifically into the lac operator at two sites. These sites within d(ApApTpTpGpTpGpApGpCpGpGpApTpApApCpApApTpT), segment I, and d(ApApTpTpGpTpTpApTpCpCpGpCpTpCpApCpApApTpT), segment II, are indicated by p. Each segment containing a chiral methylphosphonate was annealed to the complementary unmodified segment. The interactions of these four modified lac operators with lac repressor were analyzed by the nitrocellulose filter binding assay. Introduction of either chiral phosphonate in segment II had little effect on the stability of the repressor-operator complex. When methylphosphonates were introduced into segment I, the affinity of lac repressor for the modified operators was shown to be dependent on the stereochemical configuration of the methylphosphonate.     

Letters to the editor: Nuclear magnetic resonance studies of protein-RNA interactions. I. The elongation factor Tu-GTP aminoacyl-tRNA complex

The 300 MHz high-resolution nuclear magnetic resonance spectra of hydrogen-bonded protons in Escherichia coli tRNA^Glu and yeast tRNA^Phe have previously been reported and the resolved resonances assigned to specific base-pairs. Here we show that in complexes of these two tRNAs with elongation factor Tu there is no discernible loss of base-paired protons. Within the experimental accuracy this means that no helical arms open upon complex formation.     

Nuclear magnetic resonance spectra of lipoteichoic acid

Lipoteichoic acid acids with a range of chemical compositions have been studied using 1H; 13C- and 31P-nuclear magnetic resonance. Proton spectroscopy provided a rapid method for demonstrating whether alanine in a sample is covalently bound to the polyglycerophosphate chains and for monitoring hydrolysis of alanine. The nature of sugar substituents can be determined, with some limitations, from the 13C spectra, and the proportions of glycerol residues substituted by alanine and sugar can be measured. The 31P spectra of lipoteichoic acid provided information about both the degree of substitution and the distribution of the substituent along the polyglycerophosphate chain, except when the substituent was galactose. The polyglycerophosphate chains were shown to undergo rapid internal rotation and no evidence for tertiary structure was found either in the presence or absence of magnesium ions. Magnesium ions exchange rapidly between the bound and free state and the binding constant to lipoteichoic acid of 64 M-1 is typical for monophosphates in aqueous solution. There was no evidence that alanine substitution affects the binding constant for magnesium ions.     

NUCPLOT: a program to generate schematic diagrams of protein-nucleic acid interactions.

Proteins that bind to DNA are found in all areas of genetic activity within the cell. To help understand how these proteins perform their various functions, it is useful to analyse which residues are involved in binding to the DNA and how they interact with the bases and sugar-phosphate backbone of nucleic acids. Here we describe a program called NUCPLOT which can automatically identify these interactions from the 3D atomic coordinates of the complex from a PDB file and generate a plot that shows all the interactions in a schematic manner. The program produces a PostScript output file representing hydrogen, van der Waals and covalent bonds between the protein and the DNA. The resulting diagram is both clear and simple and allows immediate identification of important interactions within the structure. It also facilitates comparison of binding found in different structures. NUCPLOT is a completely automatic program, which can be used for any protein-DNA complex and will also work for certain protein-RNA structures.     

An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli

The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is ‘orthogonal’, meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA^Glu sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.