Identification of a selenocysteine-specific aminoacyl transfer RNA from rat liver

The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [⁷⁵Se]selenocysteine and [⁷⁵Se]selenite as substrates. [⁷⁵Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [⁷⁵Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [³⁵S]cysteme, the most highly purified ⁷⁵Se-fractions were > 100-fold purified relative to ³⁵S. These fractions contained < 0.7% of the [³⁵S]cysteine originally present in the total tRNA. When [³⁵Se]selenocysteyl tRNA was purified from a mixture of ¹⁴C-labeled amino acids, over 97% of the [¹⁴C]aminoacyl tRNA was removed. The [⁷⁵Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [⁷⁵Se]selenocysteine, reaction with hydroxylamine and recovery of [⁷⁵Se]selenocysteyl hydroxamic acid and release of ⁷⁵Se by ribonuclease. The specificity of [⁷⁵Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.     

Evidence for interaction of an aminoacyl transfer RNA synthetase with a region important for the identity of its cognate transfer RNA

Recent experiments showed that a single base pair (G3:U70) in the amino acid acceptor helix is a major determinant for the identity of Escherichia coli alanine transfer RNA. Experiments reported here show that bound alanine tRNA synthetase protects (from ribonuclease attack) seven consecutive phosphodiester linkages on the 3'-side of the acceptor-T psi C helix (phosphates 65-71) and a few additional sites that are in scattered locations. There is no evidence for interaction of the enzyme with the anticodon, a sequence which can be varied without effect on recognition by alanine tRNA synthetase.     

Release of aminoacyl transfer ribonucleic acid from transfer ribonucleic acid synthetase studied by rapid molecular sieve chromatography

Complex of Ile-tRNA^Ile and isoleucyl-tRNA synthetase (IRS) was isolated by rapid chromatography on a Bio-Gel P-100 column at 4°C. By incubating the complex in the presence of excess unacylated tRNA^Ile prior to chromatography, it is possible to qualitatively measure the rate of exchange of Ile-tRNA^Ile with tRNA^Ile on the enzyme. The rate of exchange is markedly accelerated by isoleucine and isoleucinyl-AMP, but not by ATP. These results confirm previously published findings that the rate of release of newly synthesized Ile-tRNA^Ile from IRS is very slow in the absence of isoleucine or isoleucyl-AMP, but that the release is greatly enhanced by these ligands. The rapid chromatography procedure thus provides a very direct and straightforward means for measuring the dynamics of a proteinnucleic acid interaction.     

Equilibrium measurements of cognate and noncognate interactions between aminoacyl transfer RNA synthetases and transfer RNA.

The interaction of Escherichia coli isoleucyl-tRNA synthetase with its cognate and five noncognate tRNAs, and of yeast valyl-tRNA synthetase with its cognate and four noncognate tRNAs, has been measured directly by fluorescence quenching. The cognate associations are strongest (association constant of 10(8) M-1 or more at pH 5.5, 17 degrees). A wide variation is found in the strengths of the noncognate interactions; these have association constants smaller than that of these cognate association by a factor of less than 10 to over 10(4), depending on the enzyme-t-RNA pair. A more detailed study of the cognate isoleucyl-tRNA synthetase-tRNAIle association suggests that the strength of the interaction is markedly sensitive to a pH-dependent transition in the enzyme centered at pH 6 on the other hand, Mg2+-induced structural changes in tRNAIle at 17 degrees in low salt do not greatly affect the availability of the nucleic acid's receptor sites for enzyme...     

Recent results on how aminoacyl transfer RNA synthetases recognize specific transfer RNAs

Summary Aminoacyl tRNA synthetases discriminate between tRNA species by a highly specific mechanism. Physical and chemical studies indicate that the synthetases bind along and around the inside of the three-dimensional L-shaped tRNA structure. Studies of mutant tRNAs that affect synthetase interaction tend to confirm this conclusion. However, in contrast to proteins that recognize a specific block of contiguous nucleotide units (e.g., repressors, restriction enzymes, etc.), synthetases appear to interact with spatially disperse elements of the structure. Available evidence suggests that tRNA binding clefts on various synthetases may be roughly similar, with specificity being achieved by the choice of amino acid residues in a few critical positions in the tRNA binding clefts. With this idea in mind, it should be possible to introduce amino acid substitutions into the binding clefts and thereby change tRNA recognition specificity. This has been attempted (by genetic manipulations) and a mutant alanine tRNA synthetase with altered tRNA recognition has been isolated. This enzyme can attach alanine to isoleucine specific tRNA. When presented with valine specific tRNA, a tRNA similar in some structural features to the isoleucine specific tRNA, or with the structurally quite different tyrosine specific tRNA, no significant aminoacylation occurs. Thus, a precise specificity alteration can occur through mutation; this result supports the idea of similarities in synthetase binding clefts, with specificity being achieved by the positioning of amino acids at critical positions in these clefts. Finally, further data have been obtained on the issue of possible transient covalent bond formation between synthetases and tRNAs, as a critical part of the interaction.Abbreviations tRNA^x a tRNA specific for the amino acid - x where x is given the standard 3 letter abbreviation     

Structural organization of complexes of transfer RNAs with aminoacyl transfer RNA synthetases.

A variety of experimental data on synthetase-tRNA interactions are examined. Although these data previously had no direct explanation when viewed only in terms of the tRNA cloverleaf diagram, they can be rationalized according to a simple proposal that takes account of the three dimensional structure of tRNA. It is proposed that a major part of the binding site for most or all synthetases is along and around the diagonal side of the tRNA structure, which contains the acceptor stem, dihydrouridine stem, and anticodon. This side of the tRNA molecule contains structural features likely to be common for all tRNAs. Depending on the system, an enzyme may span a small part or all of the region of this side of the molecule. Interactions with other parts of the structure may also occur in a manner that varies from complex to complex. These interactions may be determined, in part, by the angle at which the diagonal side of the flat tRNA molecule is inserted onto the surface of the synthetase.     

Interrelation between transfer RNA and amino-acid-activating sites of methionyl transfer RNA synthetase from Escherichia coli.

Binding of tRNA(Met/f) to the monomeric trypsin-modified methionyl-tRNA synthetase turns off the methionine-dependent isotopic ATP--PPi exchange. In the case of the dimeric native methionyltRNA synthetase, one anticooperatively bound tRNA(Met/f) inhibits the exchange by only 50%. These behaviours of tRNA do not require the integrity of the 3'-terminal adenosine. Esterification by methionine of the 3' end of tRNA reinforces the affinity of tRNA(Met/f)for the enzymes. In the case of the native enzyme, due to this effect, a second binding mode for methionyl-tRNA may be demonstrated through the isotopic exchange. This additional binding of tRNA corresponds to the expression of the anticooperatively blocked tRNA binding site. Methionine reverses competitively the reinforcing effect of the esterified methionyl moiety on tRNA binding. It is concluded that after esterification of tRNA, the aminoacyl residue still binds the enzyme, probably within the methionine activating site. The latter behaviour may account for the observation that excess methionine accelerates the aminoacylation turnover rate of tRNA(Met/f).