RESEARCH
tRNA activity depends on unique conformation Physical chemical comparison of active and inactive forms of a leucine-acceptor tRNA reveals structural differences Strong evidence has accumulated at Princeton University that the important, remarkably specific behavior of transfer ribonucleic acid (tRNA) in the biological assembly of proteins derives from specific stereochemical arrangements of the tRNA molecule. These latest indications, from the laboratory of Dr. J. R. Fresco of Princeton, rule out unrestricted mobility of tRNA conformations. The results also raise the question of the exact source of the amino acid carrier's conformational specificity. The problem is whether specificity resides in primary structure alone (nucleotide sequence) or, instead, includes dependence on secondary structure (helical formations) and tertiary structure (weak interactions of unpaired residues which fix helical formations in space). Most recently, the work by Dr. Fresco and his associates, Dr. Tomas Lindahl and Alice Adams, in Princeton's biochemical sciences program, has demonstrated major conformational differences between two forms of a leucine-specific tRNA isolated from baker's yeast [Proc. Natl. Acad. Set. U.S., 57, 1684 (1967)]. These results follow the discovery last
year by the same investigators, and simultaneously by Princeton's Dr. Noboru Sueoka and William Gartland, that certain tRNA molecules from yeast and Escherichia coli can be trapped in biologically inactive forms. One of the two forms in the present work is the normal, biologically active tRNA, called "native." Dr. Fresco and his associates term the other form "denatured," since it shows little or no activity as a substrate in a variety of biosynthetic reactions catalyzed by enzymes associated with protein synthesis. The denatured tRNA can be kept in this state for a relatively long time in the presence of 0.005 to 0.02M Mg 2 +. Renaturation to the native state merely requires heating to 60° C. for five minutes. Calculations from viscosity and sedimentation data give molecular weights of 29,800 ± 1500 for native leucine-specific tRNA and 30,600 ± 1500 for the denatured form. The difference in the two values of less than 3 % shows that the denatured form is not a dimer or higher aggregate. Gross structural differences between the two tRNA states include a
2 5 % greater hydrodynamic sphere (deduced from viscosity and sedimentation data) for the denatured form. The denatured molecule is thus somewhat more open (less coiled) or swollen, and perhaps more asymmetric than the native form. However, the difference is still small in comparison to the threefold viscosity increase shown by the native tRNA when heated to 85° C. At this high temperature, the viscosity reaches values typical of a completely unfolded polyelectrolyte such as polyuridylic acid. Still, this molecular enlargement leading to the denatured state undoubtedly reflects a major change in tertiary structure, Dr. Fresco and his associates conclude. The change is consistent with a striking increase in the denatured molecule's sensitivity to attack by pancreatic ribonuclease and other enzymes. The change is also consistent with their previous finding, by more direct physical chemical means, of tertiary structure in tRNA. Gel filtration behavior of the two tRNA forms also bears out their differing molecular dimensions. The two forms were filtered through columns of Sephadex G-100 as solutions
Optical and gel filtration properties show structural differences between native and denatured
1000 5
Effluent, milliliters
Filtration through columns of Sephadex G-100 gives distinct peaks for denatured and native forms of leucine-acceptor tRNA (squares) and tagged native 14C-leucine-tRNA (triangles) and denatured 3H-leucine-tRNA (circles) 34 C&EN JULY 17, 1967
Wave length, millimicrons
Optical rotatory dispersion of leucine-acceptor tRNA in two conformations at 25° C. shows red shift in the crossover wave length of the Cotton effect. Shift indicates loss of base pairs in secondary structure of denatured form
in the magnesium-containing solvent, 0.15M KCl + 0.01M K-cacodylate + 0.005M MgCl 2 + 0.0005M EDTA (pH = 7.0). This solvent was used for all physical measurements in this study. In the filtration, native leucinespecific tRNA is displaced to the ascending side of the main peak of eluted, unfractionated native tRNA. When a mixture of the two forms of this leucine-acceptor tRNA in a purified state is similarly filtered, denatured leucine-specific tRNA is much less retarded than the native form. This is consistent with its more expanded or asymmetric structure. It was by taking advantage of the lesser retardation of the denatured form that the Princeton chemists first obtained this leucine-acceptor tRNA in pure form. The filtration technique is also applicable for partial purification of some other amino acidspecific tRNA's that can be trapped in denatured form [/. Biol, Chem., 242, 3129 (1967)]. Additional variations occur in the secondary structure of the two tRNA conformations. Ultraviolet absorption spectra indicate a reduction of about 10% in helical content for the denatured structure. The spectral difference corresponds to a loss of two to three adenine-uracil (A-U) and one guanine-cytosine (G-C) WatsonCrick-type base pairs. Remaining helical content would have the same proportion of A-U and G-C nucleotide pairs as the native form. Other evidence for the denatured state's reduced secondary structure comes from optical rotation and circular dichroism measurements made
with a spectropolarimeter and dichrograph. Conversion of the native to denatured tRNA results in a red shift in optical rotation data. The shift is from 243 to 252 m/x for the one-term Drude dispersion constant and from 260.8 to 263.3 m^ for the crossover wave length of the Cotton effect. A similar red shift shows up in the maximum of the circular dichroism spectrum. The red shift indicates that some base pairs in the molecules have been disrupted to form single-stranded (unpaired) nucleotides. The net decrease, as in the optical absorbance data, is small—about three to four base pairs equally divided between A-U and G-C combinations. In spite of this small apparent change in base pairs, Dr. Fresco and his associates point out that conversion of the denatured to the native state involves a radical structural disruption and reformation, not merely superposition of tertiary structure on nearly completed secondary structure. The high activation energy barrier between the two tRNA states (now measured at Princeton to be 60 kcal.) bears out the extent of this change. Renaturation involves, in effect, disruption of a "wrong" secondary and tertiary arrangement followed by formation of a "correct" one. Thus, Dr. Fresco and his associates note, tRNA exhibits a dependence of activity on macromolecular structure just as do enzymes, which are globular proteins with catalytic activity. These enzymes' structural dependence has been recognized for some time. The logical extension of the analogy between tRNA and globular proteins,
leucine-acceptor transfer ribonucleic acid
in Dr. Fresco's view, is that tRNA molecules, like proteins, should be crystallizable. Obtainment of crystals might then afford a more classical chemical approach to linking biological specificity of tRNA in protein synthesis to macromolecular structure. A tRNA imparts specificity to protein synthesis at two levels. One involves adapting a particular amino acid to a form (by bonding the acid to the tRNA) which can respond precisely to genetic code directions. The other involves reading the coded message precisely (on the messenger RNA) thereby locating the amino acid at the proper locus in a growing polypeptide chain. Elucidation of tRNA structure also requires identification of the nucleotide sequence making up the molecule's primary structure. First such elucidation, in 1965, was for alaninespecific tRNA by Dr. Robert W. Holley and a team at Cornell University and the U.S. Department of Agriculture's Plant, Soil, and Nutrition Laboratory, Ithaca, N.Y. Sequences of at least five other tRNA's are known (C&EN, June 5, page 46). Yeastderived tRNA's now deciphered, for example, include those for serine, valine, alanine, and phenylalanine.
RESEARCH IN BRIEF A black carpet beetle attractant has
been identified by Dr. R. M. Silverstein and J. O. Rodin of Stanford Research Institute, Menlo Park, Calif. Assisted by Dr. W. E. Burkholder and J. E. Gorman of Agricultural Research Service, the Stanford Research scientists have isolated, identified, and synthesized the attractant under a USDA contract. The substance is a carboxylic acid, frans-3,ds-5-tetradecadienoic acid, and is a sex attractant for the male black carpet beetle, Attagenus piceus. A common pest, the beetle may be controlled by the males' response to the synthetic attractant. The oxidation of cobalt-chromium alloys is to be studied at Battelle Memorial Institute, Columbus, Ohio, under a NASA research grant. Cobaltchromium alloys are used in high-temperature, oxidizing systems such as jet engines. The objective of the oneyear program, headed by Dr. R. I. Jaffee, is an understanding of basic oxidation behavior at 1400° to 2400° F. using alloys containing 10, 25, and 35% chromium.
Time, minutes
Denatured leucine-acceptor tRNA shows much more sensitivity (from more available sensitive sites) to attack by pancreatic ribonuclease than does the native form. Reaction degrades tRNA forms to acid-soluble fragments
The correct journal reference to Ko-
rad Corp.'s work on organic dye lasers is Appl. Phys. Letters, 10, 266 (1967), not Phys. Rev. Letters, as given in C&EN, June 19, page 38. JULY 17, 1967 C & E N
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