The aminoacylation specificity ("acceptor identity") of transfer RNAs (tRNAs) has previously been associated with the position of particular nucleotides, as opposed to distinctive elements of three-dimensional structure. The contribution of a G⋅U wobble pair in the acceptor helix of tRNAAla to acceptor identity was examined with synthetic amber suppressor tRNAs in Escherichia coli. The acceptor identity was not affected by replacing the G⋅U wobble pair in tRNAAla with a G⋅A, C⋅A, or U⋅U wobble pair. Furthermore, a tRNAAla acceptor identity was conferred on tRNALys when the same site in the acceptor helix was replaced with any of several wobble pairs. Additional data with tRNAAla show that a substantial acceptor identity was retained when the G⋅U wobble pair was translocated to another site in the acceptor helix. These results suggest that the G⋅U wobble pair induces an irregularity in the acceptor helix of tRNAAla to match a complementary structure in the aminoacylating enzyme.
The specificity of tRNA(Arg) (arginine transfer RNA) for aminoacylation (its acceptor identity) were first identified by computer analysis and then examined with amber suppressor tRNAs in Escherichia coli. On replacing two nucleotides in tRNA(Phe) (phenylalanine transfer RNA) with the corresponding nucleotides from tRNA(Arg), the acceptor identity of the resulting tRNA was changed to that of tRNA(Arg). The nucleotides used in the identity transformation occupy a "variable pocket" structure on the surface of the tRNA molecule where two single-stranded loop segments interact. The middle nucleotide in the anticodon also probably contributes to the interaction, since an amber suppressor of tRNA(Arg) had an acceptor identity for lysine as well as arginine.
The diverse and highly specific interaction between RNAs and proteins plays an essential role in many important biological processes. In the glutamine aminoacylation system, crystal structures of the free and ligated macromolecules have provided a description of the tRNA-protein interactions at the molecular level. This data lays the foundation for genetic, biochemical, and structural analyses to delineate the set of key interactions that governs the structure-function relationships of the two macromolecules. To this end the chromosomal tRNA(Gln) genes were disrupted in Escherichia coli to produce a tRNA(Gln) knockout strain that depends upon expression of a functional tRNA(Gln) from a plasmid for cell viability. Mutants of an inactive tester tRNA derived from tRNA(Ala) were generated by hydroxylamine mutagenesis, and the active derivatives were selected by their ability to support knockout cell growth. Two of the mutants contained substitutions in the first base pair of the acceptor stem that likely facilitate the formation of a hairpin loop that places A76 in the active site. The third mutation was located at position 13 in the D loop region of the tRNA, and suggests that an interaction with residue 13 contributes to a specific conformational change in unliganded GlnRS, which helps configure the enzyme active site in its catalytically proficient form. This work demonstrates the efficacy of an integrated approach that combines genetic selections and biochemical analyses with the physical data from crystal structures to reveal molecular steps that control the specificity of RNA-protein interactions.
Previous studies indicated that (i) T4 gene s product (gps) protects infected cells from superinfection lysis from without, (ii) the absence of gps in infected cells also leads to lysis from within even when T4 e lysozyme is absent, (iii) T4 gene 5 product (gp5), a polypeptide of the virion baseplate, may be responsible for inducing lysis from without, and (iv) altered gp5 of the T4 mutant 5ts1 can replace e lysozyme to cause lysis from within. Results of this study showed that (i) wild-type gp5 in infected cells lacking e lysozyme was responsible for lysis from within in the absence of gps, and (ii) gps did not protect infected cells from superinfection lysis from without by 5ts1 phage. We prpose that gps normally prevents functional expression of wild-type gp5 activity from either side of the cell wall, whereas the 5ts1 form of gp5 is insensitive to the gps barrier and induces lysis from either side of the cell wall.
The structural features that determine tRNA(Ala) acceptor identity have been studied with amber-suppressor tRNAs in Escherichia coli cells. Previous work established that a wobble pair composed of guanosine at position 3 and uridine at position 70 (G3-U70) in the acceptor helix of tRNA(Ala) is a determinant of the molecule's acceptor identity. We show that additional determinants are located at three other sites in the acceptor helix and at one site in the variable pocket of tRNA(Ala). These latter determinants are less important than G3.U70 since their individual alterations in mutants of tRNA(Ala) have smaller degrading effects on the functions of the molecules, and subsets of the determinants, when combined with G3.U70, are sufficient to switch the identities of several other tRNAs to that of tRNA(Ala). Other workers are using fragments of the tRNA(Ala) acceptor helix to study the molecule's acceptor identity. Our demonstration that the variable pocket contributes to tRNA(Ala) acceptor identity means that such fragments do not faithfully replicate the structure-function relationship of the cellular process.
