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.

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

The complex formation between the single-strand DNA binding protein (ssB protein) from Escherichia coli and oligonucleotides and single-stranded DNA has been studied by using fluorescence titrations, ultracentrifugation measurements, and fast kinetic techniques. Determination of the stoichiometries of oligo(dT)--ssB complexes shows that each of the four subunits of the ssB protein represents a binding site for an oligonucleotide about eight residues long. Occupation of all four binding sites with oligo(dT) or poly(dT) leads to 80% quenching of the intrinsic protein fluorescence. The binding sites are nearly equivalent and independent. For d(pT)16, the intrinsic binding constant is 6 X 10(5) M-1, and for d(pT)30-40, which is long enough to extend continuously over the ssB tetramer, the binding constant is higher than 5 X 10(8) M-1. Oligoadenylates bind about 2 orders of magnitude weaker than the corresponding oligo(dT) species. The binding of oligo(dT) is very weakly dependent on ionic strength, in contrast to the oligo(dA)--ssB complex formation. For d(pT)8, d(pT)16, and d(pT)30-40, the complex formation can be described by a simple one-step reaction. The strength of the interaction is mainly expressed in the rate constant of dissociation. In the cooperative complexes with poly(dT) or poly(dA), all four binding sites on the ssB tetramer are also occupied. It is concluded that single-stranded DNA is coiled around the ssB molecule. Fluorescence melting experiments of the complexes show that the conformation of the single-stranded DNA has a strong influence on the stability of the complexes.

Escherichia coli single-strand deoxyribonucleic acid binding protein: stability, specificity, and kinetics of complexes with oligonucleotides and deoxyribonucleic acid

The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNA1Val (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10766.x

Mechanism of Discrimination between Cognate and Non‐cognate tRNs by Phenylalanyl‐tRNA:Synthetase from Yeast

Thermodynamic analysis of transfer RNA unfolding.

Kinetics of conformational changes in tRNAPhe (yeast) as studied by the fluorescence of the y-base and of formycin substituted for the 3'-terminal adenine

Cad R. Rabl

Papers

Kinetics of conformational changes in tRNAPhe (yeast) as studied by the fluorescence of the y-base and of formycin substituted for the 3'-terminal adenine