Tryptophanyl-tRNA synthetase from Bacillus subtilis. Characterization and role of hydrophobicity in substrate recognition.

The tryptophanyl-tRNA synthetase from Bacillus subtilis was purified to homogeneity and characterized. It has an alpha 2 subunit structure and a molecular weight of 77,000. Tryptophanyl-tRNA synthetase does not catalyze any significant proofreading. It activates tryptophan as well as the three fluorinated analogues, DL-4-fluoro-, DL-5-fluoro-, or DL-6-fluorotryptophan (4F-, 5F-, and 6F-Trp), in the ATP-pyrophosphate exchange reaction. In the aminoacylation reaction, the fluorotryptophans act as competitive inhibitors of Trp. Their relative activities follow the same order in both reactions: Trp greater than 4F-Trp greater than 6F-Trp greater than 5F-Trp. This order is the inverse of the order of relative hydrophobicities of these compounds, pointing to the importance of hydrophobic interactions in the selective recognition by tryptophanyl-tRNA synthetase among this group of substrates. To define the physical basis of the relative hydrophobicities, the crystallographic structure of 4F-Trp was determined and compared to that of trptophan. Charge distributions calculated for tryptophan and its different fluoroanalogues on the basis of molecular structures were supported by their carbon-13 NMR spectra. Correlations between charge distributions and relative hydrophobicities suggest that the polarity of the C-F bond represents an underlying factor determining the hydrophobicities of 4F-, 5F-, and 6F-Trp, thus relating tryptophanyl-tRNA synthetase selectivity toward tryptophan and its fluoroanalogues directly to their electronic configurations.

Suppression of fluorescence of tryptophan residues in proteins by replacement with 4-fluorotryptophan.

The tryptophan-auxotrophic Bacillus subtilis LC33 mutant strain utilizes either tryptophan or 4-fluorotryptophan for growth. Proteins therefore could be isolated from these cells in either tryptophan-containing or 4-fluorotryptophan-containing forms. Since 4-fluorotryptophan is non-fluorescent, tryptophan fluorescence would be suppressed in the 4-fluorotryptophan-containing proteins, facilitating the investigation of other chromophores either on the proteins or interacting with the proteins. This approach, potentially applicable to any protein endogenous to or clonable into B. subtilis, was illustrated by an examination of the fluorescence of B. subtilis ribosomal proteins.

Complementary base-pairing properties of cyclized and linear oligonucleotides.

Oligouridylates of varying chain lengths were synthesized by polynucleotide phosphorylase and cyclized by RNA ligase. Over chain lengths from 7 to 15, the bindings of the cyclized and linear oligomers to polyadenylate were measured on the basis of differential migration of bound and free oligomers on a gel exclusion column. Binding of the cyclized oligomers was found to be far weaker than that of their linear counterparts of equal length. Such a general reduction in base-pairing capacity due to the cyclized conformation, by limiting the strength of unintended base-pairing without obstructing the possible development of strong specific base-pairing, may represent an advantage important to the function and evolution of loop structures in tRNA and other RNA molecules.

Inadequacy of prebiotic synthesis as origin of proteinous amino acids.

The production of some nonproteinous, and lack of production of other proteinous, amino acids in model prebiotic synthesis, along with the instability of glutamine and asparagine, suggest that not all of the 20 present day proteinous amino acids gained entry into proteins directly from the primordial soup. Instead, a process of active co-evolution of the genetic code and its constituent amino acids would have to precede the final selection of these proteinous amono acids.

Low-affinity interaction of ethanol with the fluorescent complex between horse liver alcohol dehydrogenase and auramine O.

Quenching of the fluorescence of the complex between horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase (EC 1.1.1.1) and auramine O complex is inconsistent with a simple competitive displacement of auramine O by ethanol. Instead, the action of ethanol requires an explanation in terms of a solvent effect, or the formation of an enzyme-auramine O-ethanol ternary complex. The latter complex would have to be the low-affinity variety similar to the enzyme-NADH-ethanol ternary complex encountered in the kinetic system.

Nature of ribosome-bound beta-galactosidase.

Properties of the ribosome-bound beta-galactosidase were examined in Escherichia coli cells after prolonged induction. This fraction of enzyme was not chased from ribosomes by removal of inducer, or by treatments with hydroxylamine, puromycin, chloramphenicol, and azide. However, the metabolic turnover of this fraction could be demonstrated by means of a pulsed exposure to the phenylalanine analogue beta-2-thienylalanine, and this fraction was enriched in heavy forms relative to the soluble enzyme. These observations indicated a tight coupling of the release of ribosome-bound enzyme to nascent enzyme synthesis, and it is suggested that the ribosome-bound enzyme is related to an intermediate stage in the assembly of quarternary enzyme structures.