Small Angle X-Ray Scattering

Cyclodextrins are cyclic oligomers of glucose that can form water-soluble inclusion complexes with small molecules and portions of large compounds. These biocompatible, cyclic oligosaccharides do not elicit immune responses and have low toxicities in animals and humans. Cyclodextrins are used in pharmaceutical applications for numerous purposes, including improving the bioavailability of drugs. Current cyclodextrin-based therapeutics are described and possible future applications discussed. Cyclodextrin-containing polymers are reviewed and their use in drug delivery presented. Of specific interest is the use of cyclodextrin-containing polymers to provide unique capabilities for the delivery of nucleic acids.

Cyclodextrin-based pharmaceutics: past, present and future

Cyclodextrins and their pharmaceutical applications.

Aminoacyl-tRNAs are substrates for translation and are pivotal in determining how the genetic code is interpreted as amino acids. The function of aminoacyl-tRNA synthesis is to precisely match amino acids with tRNAs containing the corresponding anticodon. This is primarily achieved by the direct attachment of an amino acid to the corresponding tRNA by an aminoacyl-tRNA synthetase, although intrinsic proofreading and extrinsic editing are also essential in several cases. Recent studies of aminoacyl-tRNA synthesis, mainly prompted by the advent of whole genome sequencing and the availability of a vast body of structural data, have led to an expanded and more detailed picture of how aminoacyl-tRNAs are synthesized. This article reviews current knowledge of the biochemical, structural, and evolutionary facets of aminoacyl-tRNA synthesis.

Aminoacyl-tRNA synthesis.

The aminoacyl-transfer RNA synthetases (aaRS) catalyse the attachment of an amino acid to its cognate transfer RNA molecule in a highly specific two-step reaction. These proteins differ widely in size and oligomeric state, and have limited sequence homology. Out of the 18 known aaRS, only 9 referred to as class I synthetases (GlnRS, TyrRS, MetRS, GluRS, ArgRS, ValRS, IleRS, LeuRS, TrpRS), display two short common consensus sequences ('HIGH' and 'KMSKS') which indicate, as observed in three crystal structures, the presence of a structural domain (the Rossman fold) that binds ATP. We report here the sequence of Escherichia coli ProRS, a dimer of relative molecular mass 127,402, which is homologous to both ThrRS and SerRS. These three latter aaRS share three new sequence motifs with AspRS, AsnRS, LysRS, HisRS and the beta subunit of PheRS. These three motifs (motifs 1, 2 and 3), in a search through the entire data bank, proved to be specific for this set of aaRS (referred to as class II). Class II may also contain AlaRS and GlyRS, because these sequences have a typical motif 3. Surprisingly, this partition of aaRS in two classes is found to be strongly correlated on the functional level with the acylation occurring either on the 2' OH (class I) or 3' OH (class II) of the ribose of the last nucleotide of tRNA.

Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs

Publisher Summary This chapter discusses that the adaptor role of tRNA requires that it has at least two functionally different sites-one for the specific interaction with the messenger, and one for accepting and transferring the particular amino acid to the growing polypeptide chain. The codon recognition mechanism of tRNA is explained in principle by the codon–anticodon interaction, including the wobble hypothesis, and has been confirmed by sequencing many tRNAs. The second function discusses that apart from the long-known fact that the -N-C-C-A end, common to all tRNAs, carries the amino acid in ester linkage, the problem of the specific attachment of an amino acid to its corresponding tRNA (the “recognition” problem) is still not solved. The chapter reviews that the -N-C-C-A end is not responsible for the specificity of the attachment; there must be other features in the molecule or in the enzymic mechanism of aminoacylation responsible for this high degree of specificity. However, these characteristics, whatever they are, must be seen in relation to the -N-C-C-A end, since this part of the tRNA carries the amino acid. Thus the presence of an intact -N-C-C-A end is a general prerequisite for the aminoacylation and for amino-acid transfer. It discusses the structural and the chemical requirements for the functioning of the -N-C-C-A terminus. The chapter summarizes recent results on the function of the -N-C-C-A end of tRNA as a reactive site during aminoacylation and ribosomal protein biosynthesis, with information about the structure of -N-C-C-A end and changes in its function after its modification.

The -C-C-A end of tRNA and its role in protein biosynthesis.

From scale models of the cyclodextrins it would appear that the primary hydroxyl groups (one per glucose unit; all glucose units c~1-+4 linked [3]) always line the outer brim irrespective of the conformation [7]. If all the glucose residues in cyclodextrins are present in the C -1 chair form [9] as NMR studies indicate [8], the CH-groups of carbons 3 and 5 of each unit and less prominently the glycosidic oxygen would form the inside of the ring [4 I. Therefore, the void space should be relatively hydrophobic. In aqueous solution each individual cyclodextrin molecule is surrounded by solvating water which in principle can be of two different types:

Inclusion Compounds of Cyclodextrins

A method is presented by which the site of primary attachment of the amino acids with respect to the 2'- or 3'-hydroxyl group of the terminal adenosine of E. coli tRNAs can be determined. It is found that the aminoacyl-tRNA synthetases (EC 6.1.1.-) with specificity for Arg, Asn, Ile, Leu, Met, Phe, Thr, Trp, and Val attach the amino acid to the 2'-position; those with specificity for Gly, His, Lys, and Ser attach the amino acid to the 3'-position; and that Tyr and Cys can be enzymatically attached to both the 2'- and 3'-positions. Together with previous experiments on yeast aminoacyl-tRNA synthetases, it is now shown that the specificity for one particular hydroxyl group is preserved during the evolution from prokaryotic to eukaryotic systems.

Site of aminoacylation of tRNAs from Escherichia coli with respect to the 2'- or 3'-hydroxyl group of the terminal adenosine.

Structural analogues of adenosine 5′-triphosphate and cytidine 5′-triphosphate were investigated as substrates for ATP(CTP): tRNA nucleotidyl transferase. Eight out of 26 ATP analogues and six out of nine CTP analogues were incorporated into the 3′ terminus of tRNA. In general, for the recognition of the substrates the modification of the cytidine is less critical than is the modification of adenosine. An isosteric substitution on the ribose residue is possible in both CTP and ATP. The free hydroxyls of these triphosphates can be replaced by an amino group or hydrogen atom without loss of substrate properties. Modifications of positions 1, 2, 6 and 8 on the adenine ring of ATP are not allowed whereas modification on positions 2, 4 and 5 on the cytosine ring of CTP are tolerated by the enzyme. No differences can be observed in the substrate properties of ATP(CTP): tRNA nucleotidyl transferase isolated from different sources. Methods for preparation of tRNA species, which are shortened at their 3′ end by one or more nucleotides, and analytical procedures for characterisation of these modified tRNAs are described.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1977.tb11985.x

Enzymatic Incorporation of ATP and CTP Analogues into the 3′ End of tRNA

Publisher Summary This chapter explores that correlation between molecular structure and biological function is one of the main aims of present day biology. The detailed mechanism of spelling out the genetic message is closely linked to subcellular structures, and molecular tools of the utmost refinement are required. The chapter reviews that tRNA's play the key role in the transmission of genetic information from DNA into protein structure. During these processes, tRNA's are specifically charged with their appropriate amino acids by enzymes that can act only on well-defined, undenatured, three-dimensional structures. tRNA's further interact with the well-defined binding sites of the ribosomal subunits. They interact with the properly aligned messenger. They carry amino acids and peptides into their correct respective positions for the formation of peptide bonds. All these highly specific nucleic acid protein interactions and nucleic acid-nucleic acid interactions cannot be understood without the existence of a well-defined three-dimensional structure of tRNA. It also provides an overview on the three independent approaches, that is, small-angle X-ray scattering, viscosity measurement, and enzymatic degradation. It concludes that tRNA has a structure of higher order that is more compact and more stable than a loose combination of helical segments. There is a tertiary structure for tRNA.

Friedrich Cramer

Papers

The -C-C-A end of tRNA and its role in protein biosynthesis.

Inclusion Compounds of Cyclodextrins

Site of aminoacylation of tRNAs from Escherichia coli with respect to the 2'- or 3'-hydroxyl group of the terminal adenosine.

Enzymatic Incorporation of ATP and CTP Analogues into the 3′ End of tRNA

Three-Dimensional Structure of tRNA