Isoleucyl transfer ribonucleic acid synthetase. Competitive inhibition with respect to transfer ribonucleic acid by blue dextran.

The inhibitory effects of blue dextran and a small dye molecule derived from it (F3GA-OH) on the steady-state reaction catalyzed by Escherichia coli isoleucy-tRNA synthetase have been studied. Blue dextran gave uncompetitive inhibition with respect to Mg.ATP, mixed inhibition with respect to L-isoleucine, and competitive inhibition with respect to tRNA. The small dye molecule (F3GA-OH) was also competitive with respect to tRNA. These inhibition patterns were not consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for aminoacyl-tRNA synthetases. They were consistent with a mechanism in which a second L-isoleucine is bound after isoleucyl-AMP synthesis and before transfer of the isoleucyl moiety to tRNA. Enzyme-bound L-isoleucine lowered the affinity of the enzyme for blue dextran approximately fivefold, a value comparable to the ninefold lowering of the enzyme's affinity for tRNA upon binding L-isoleucine. The affinity of the synthetase for F3GA-OH (K1 = 1.0 X 10(-7) M) is approximately fivefold higher than its affinity for blue dextran (K1 = 5.3 X 10(-7) M). These results indicate that blue dextran and its derivatives may be useful for kinetic and physical studies of polynucleotide binding sites on proteins as well as NAD and ATP sites.     

Three photo-cross-linked complexes of yeast phenylalanine specific transfer ribonucleic acid with aminoacyl transfer ribonucleic acid synthetases.

Yeast tRNA-Phe has been cross-linked photochemically to three aminoacyl-tRNA synthetases, yeast phenylalanyl-tRNA synthetase, Escherichia coli isoleucyl-tRNA synthetase, and E. coli valyl-tRNA synthetase. The two non-cognate enzymes are known to interact with tRNA-Phe. In each complex, three regions on the tRNA are found to cross-link. Two of these are common to all of the complexes, while the third is unique to each. Thus, the cognate and non-cognate complexes bear considerable similarity to each other in the way in which the respective enzyme orients on tRNA-Phe, a result which was also established for the complexes of E. coli tRNA-Ile (BUDZIK, G.P., LAM, S.M., SCHOEMAKER, H.J.P., and SCHIMMEL, P.R. (1975) J. Biol. Chem. 250, 4433-4439). The common regions include a piece extending from the 5'-side of the acceptor stem to the beginning of the dihydrouridine helix, and a segment running from the 3' side of the extra loop into the TpsiC helix. These two regions overlap with and include some of the homologous bases found in eight tRNAs aminoacylated by yeast phenylalanyl-tRNA synthetase (ROE, B., SIROVER, M., and DUDOCK, B. (1973) Biochemistry 12, 4146-4153). Although well separated in the primary and secondary structure, these two segments are in close proximity in the crystallographic tertiary structure. In two of the complexes, the third cross-linked fragment is near to the two common ones. The picture which emerges is that the enzymes all interact with the general area in which the two helical branches of the L-shaped tertiary structure fuse together, with additional interactions on other parts of the tRNAas well.     

Conservation of transfer ribonucleic acid and 5S ribonucleic acid cistrons in Enterobacteriaceae.

The genes for tranfer ribonucleic acid (tDNA) and 5S ribonucleic acid (5SDNA) were isolated from the total deoxyribonucleic acid (DNA) of Escherichia coli. The relatedness of tDNA and 5S from E. coli and other species of Enterobacteriaceae was determined by reassociation of the isolated genes labeled with 32PO4 to unlabeled, unfractionated DNA. Double-stranded DNA was separated from unreacted DNA by hydroxyapatite chromatography. Thermal elution profiles were done to determine the amount of unpaired bases present in related DNA sequences. Relative to total DNA, both 5S DNA and tDNA were highly conserved throughout the Enterobacteriaceae, including the genera Yersinia and Proteus.     

Effects of estradiol on uterine ribonucleic acid metabolism. Assessment of transfer ribonucleic acid methylation.

Immature rats treated with estradiol for selected periods of time demonstrated both increased methylation of uterine transfer ribonucleic acid (tRNA) and methylase activities. Whereas the former parameter was assessed by incubating whole uteri with [methyl-14C]methionine and measuring the incorporation of isotope into the tRNA, methylase activity was obtained by measuring the rate of incorporation of methyl groups from S-adenosyl[methyl-14C]methionine into heterologous tRNA (Escherichia coli B) in the presence of uterine cytosol preparations (100,000g supernatants). Although increased methylation of tRNA during the estrogen response was demonstrated, additional studies indicated that these results were largely attributable to an increased rate of synthesis of tRNA rather than gross changes in either the type or amount of methylated constituents present. Evidence in this regard included the inability of estrogen treatment of alter significantly the (a) resulting patterns of methyl-14C-methylated constituents of uterine tRNA, (b) the extent ot which [2-14C]guanine residues, incorporated into tRNA, become methylated, (c) the extent of methylation of precursor tRNA in the absence of tRNA synthesis, and (d) the types of methylase activities expressed in vitro.     

Specific spin-labeling of transfer ribonucleic acid molecules.

The spin labels anhydride (ASL), bromoacetamide (BSL) and carbodiimide (CSL) were used to label selectively tRNAGlu, tRNA fMet and tRNAPhe from E. coli. The preparation and characterization of the sites of labeling of eight new spin-labeled tRNAs are described. The sites of labeling are: s2U using ASL, BSL and CLS and tRNAGlu; s4U using ASL and BSL on tRNAfMet and tRNAPhe; U-37 with CSL on tRNfMet; U-33 with CSL on tRNAPhe. The rare base X at position 47 of tRNAPhe has been acylated with a spin-labeled N-hydroxysuccinimide (HSL). The 3'end of unfractionated tRNA molecules has been chemically modified to a morpholino spin-labeled analogue (MSL). Their respective e.s.r. spectra are reported and discussed.     

The postnatal methylation of transfer ribonucleic acid in brain. Evidence for the methylation of precursor transfer ribonucleic acid.

Incubation of 3-day-old rat brain with L-[methyl-3H]methionine resulted in the rapid labeling of low-molecular-weight cytoplasmic RNA. Electrophoresis in 15% polyacrylamide gels provided evidence for the methylation of precursor tRNA molecules, and high-performance liquid chromatography demonstrated N2-methylguanine to be the predominant methylated base formed during the first 2 min of labelling.     

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].