Nucleotide handles

The search for RNAs with new catalytic functions has been aided by the development of a method that rapidly searches pools of random polymers of RNA and extracts those with particular activities SELEX is nothing less than accelerated evolution in a test tube (Box 26-3). It has been used to generate RNA molecules that bind to amino acids, organic dyes, nucleotides, cyano-cobalamin, and other molecules. Researchers have isolated ribozymes that catalyze ester and amide bond formation, Sn2 reactions, metallation of (addition of metal ions to) porphyrins, and carbon-carbon bond formation. The evolution of enzymatic cofactors with nucleotide handles that facilitate their binding to ribozymes might have further expanded the repertoire of chemical processes available to primitive metabolic systems. 

Suppose one wanted to study a biological system, possibly as small as an amino acid or as big as a polypeptide or a small nucleotide strand. There are a number of computer programs equipped to handle such systems, including the Gaussian programs. 

The aspect to which far more attention has been directed is the nature of the inserted gene(s) and promoters in rDNA products. Again, the toxicologist should ask how well the nucleotide sequence is known, whether there is only one reading frame, and how any introns are handled. Again, toxicity-type testing would appear to be an inefficient and expensive way to study molecular biology and biochemistry. 

Many experiments have been carried out by using this setup the stretching of single DNA molecules, the unfolding of RNA molecules or proteins, and the translocation of molecular motors (Fig. 2). Here we focus our attention on force experiments where mechanical work can be exerted on the molecule and nonequilibrium fluctuations are measured. The most successful studies along this line of research are the stretching of small domain molecules such as RNA [83] or protein motifs [84]. Small RNA domains consist of a few tens of nucleotides folded into a secondary structure that is further stabilized by tertiary interactions. Because an RNA molecule is too small to be manipulated with micron-sized beads, it has to be inserted between molecular handles. These act as polymer spacers that avoid nonspecific interactions between the bead and the molecule as well as the contact between the two beads. 

CoA, the coenzyme A derivative of acetoacetate, reduces its reactivity as a substrate for /3-ketoacyl-CoA transferase (an enzyme of lipid metabolism) by a factor of 106. Although this requirement for adenosine has not been investigated in detail, it must involve the binding energy between enzyme and substrate (or cofactor) that is used both in catalysis and in stabilizing the initial enzyme-substrate complex (Chapter 6). In the case of /3-ketoacyl-CoA transferase, the nucleotide moiety of coenzyme A appears to be a binding handle that helps to pull the substrate (acetoacetyl-CoA) into the active site. Similar roles may be found for the nucleoside portion of other nucleotide cofactors. 

To handle the mass of existing data, powerful computer programs have been developed and various graphical procedures have also been developed to help the human mind comprehend the results.654 655 One important problem is to define and locate what are called consensus sequences. The problem is best illustrated by examples.654 The cleavage site for the EcoRI restriction endonuclease is GAATTC. There is no ambiguity. In a DNA of random sequence this would be expected to occur by chance in about (1 / 4)6 nucleotides (4 kb). On the other hand, the Hmll restriction endonuclease cleaves within the consensus sequence GTYRAC where Y = C or T and R = A or G. 

Artificial chemical systems capable of Darwinian evolution have also been prepared from artificial laboratory genetic systems. Such systems were created in the laboratory by using an artificial DNA that contained six nucleotide letters rather than the four in standard terran DNA.6,7 These were chosen from the structures shown in Figure 4.1. The artificial systems can support the basic elements of Darwinian evolution (reproduction, mutation, and inheritance of mutated forms) even if the enzymes that support the evolution of artificial genetic systems are the natural terran enzymes that have evolved for billions of years to handle standard nucleobases. 

Work with synthetic biology makes it clear that the core set of nucleotides and the core set of amino acids found in all terran life inspected to date are not the only nucleotides and amino acids that can function in genetic and catalytic systems. Indeed, synthetic alternatives to standard terran biochemistry function in the terran machinery that has evolved to handle the core sets. 

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