Molecular biology, biochemistry, and biophysics最新文献

UMP biosynthesis requires six enzyme activities. Five of these enzyme centers are clustered into two multienzymatic proteins which are known to, or appear to, sequester the intermediates carbamyl approximately P, carbamyl aspartate and orotidylic acid. The advantages of sequestering these intermediates appear to be a conservation of energy, since two intermediates, carbamyl approximately P and orotidylate, might otherwise be rapidly degraded in mammalian cells. Carbamyl-aspartate appears not to be degraded rapidly in mammalian cells but it can pass into the blood and could possible disrupt brain metabolism by action as an acetylaspartate analog, if it passes the blood-brain barrier. For this, and possible for other reasons, there may be advantages to the fact that these intermediates are not other reasons, there may be advantages to the fact that these intermediates are not readily released from Complex A and U. In addition, these multienzymatic proteins may have other kinetic advantages, some of which have been discussed above. Studies with intact cells illustrate that azauridine, a chemical designed originally as an antineoplastic drug, produces a "ripple" effect when it inhibits the last enzyme of this pathway which leads to a sequential accumulation of pools of the various intermediates or their metabolites. This same agent increases the amount of some of the enzymes of this biosynthetic pathway in cells exposed to this drug. Both of these effects can negate the effectiveness of this potential antineoplastic drug. Sophisticated drug design may depend on whole-cell studies, such as those discussed here, in addition to the classic studies on the inhibition of a single enzyme center to select drugs that may be without significant side effects when they are finally tested in animals.

The use of fluorescent derivatives of tRNAPhe (yeast) in studies on tRNA conformation and on tRNA-ribosome recognition is described. Evidence is presented which indicates that under physiological conditions with respect to ionic strength and Mg2+ concentration, tRNAPhe exists in at least two conformations. The functional significance of this behavior is discussed on the basis of aminoacylation experiments. The investigation of the ribosome complexes of tRNAPhe labeled in the anticodon and D-loops has provided evidence suggesting that the presence of the codon, although not appreciably altering the apparent association constant, leads to qualitatively different complexes in which the tRNA appears to be rigidly bound to the codon even in the P-tRNA to the ribosome occurs in several steps, which take place only in the presence of the proper codon. One or more of these steps may represent codon-induced conformational changes of the tRNA molecule, which constitute the molecular basis of the highly specific binding of the tRNA to the ribosome.

Ribosomes are multicomponent particles on which biosynthesis of proteins occurs in all organisms. The best-known ribosome, namely that of E. coli, consists of three RNA's and 53 different proteins. All proteins have been isolated and characterized by chemical, physical, and immunological methods. the primary sequences of 49 E. coli ribosomal proteins have so far been determined. Studies of the shape, as well as of the secondary and tertiary structure, of the proteins are in progress. Various techniques, 3.g., immune electron microscopy and cross-linking of neighboring components in situ, give information about the architecture of the ribosomal particle. The first technique resulted in illustrative and detailed knowledge now only on the shape of the ribosomal subunits but also about the location of many proteins on the surface of the particles. The analysis of cross-links between ribosomal proteins and/or RNA's has in several cases been pursued to the level of elucidating which amino acids and/or nucleotides are cross-linked together in situ. Reconstitution of a fully active E. coli 50S ribosomal subunit from its isolated RNA and protein components can be accomplished by means of a two-step incubation procedure. From the analysis of the intermediates occurring during the reconstitution process it has been concluded that the in vitro reconstitution process resembles that in vivo assembly of 50S subunits in many respects. E. coli mutants with alterations in almost all ribosomal proteins have been isolated. Their biochemical and genetic analyses are very useful tools for obtaining information about the structure, function, and biosynthesis of ribosomes, as well as about the location of the genes for these proteins on the chromosome. From comparative electrophoretic, immunological, protein-chemical, and reconstitution studies on ribosomes from various species it has become clear that their is little homology between ribosomal proteins from prokaryotes and those from eukaryotes. This finding is surprising since there is no essential difference in the way in which pro-and eukaryotic ribosomes function in protein biosynthesis.