Changing the acceptor identity of a transfer RNA by altering nucleotides in a "variable pocket".

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.

Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end.

Although the genetic code for protein was established in the 1960's, the basis for amino acid identity of transfer RNA (tRNA) has remained unknown. To investigate the identity of a tRNA, the nucleotides at three computer-identified positions in tRNAPhe (phenylalanine tRNA) were replaced with the corresponding nucleotides from tRNAAla (alanine tRNA). The identity of the resulting tRNA, when examined as an amber suppressor in Escherichia coli, was that of tRNAAla.

Model substrates for an RNA enzyme.

M1 RNA, the catalytic RNA subunit of Escherichia coli ribonuclease P, can cleave novel transfer RNA (tRNA) precursors that lack specific domains of the normal tRNA sequence. The smallest tRNA precursor that was cleaved efficiently retained only the domain of the amino acid acceptor stem and the T stem and loop. The importance of the 3' terminal CCA nucleotide residues in the processing of both novel and normal tRNA precursors implies that the same enzymatic function of M1 RNA is involved.

Functional evidence for indirect recognition of G.U in tRNA(Ala) by alanyl-tRNA synthetase.

The structural features of the G.U wobble pair in Escherichia coli alanine transfer RNA (tRNA(Ala)) that are associated with aminoacylation by alanyl-tRNA synthetase (AlaRS) were investigated in vivo for wild-type tRNA(Ala) and mutant tRNAs with G.U substitutions. tRNA(Ala) with G.U, C.A, or G.A gave similar amounts of charged tRNA(Ala) and supported viability of E. coli lacking chromosomal tRNA(Ala) genes. tRNA(Ala) with G.C was inactive. Recognition of G.U by AlaRS thus requires more than the functional groups on G.U in a regular helix and may involve detection of a helical distortion.

Association of transfer RNA acceptor identity with a helical irregularity.

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 tRNA(Ala) 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 tRNA(Ala) with a G.A, C.A, or U.U wobble pair. Furthermore, a tRNA(Ala) acceptor identity was conferred on tRNA(Lys) when the same site in the acceptor helix was replaced with any of several wobble pairs. Additional data with tRNA(Ala) 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 tRNA(Ala) to match a complementary structure in the aminoacylating enzyme.

Specific duplications fostered by a DNA structure containing adjacent inverted repeat sequences.

This paper describes the nucleotide sequences of three spontaneous mutations in a suppressor gene of phage T4 tRNA(Ser). They are duplications of the anticodon and variable arms of the tRNA(Ser) molecule. One is a 34-nucleotide direct repeat of the wild-type sequence. The remaining two have reciprocal structures, with each containing 35-nucleotide inverted and direct repeats of the wild-type sequence. One of the latter mutations is frequent and was present in multiple isolates. All three duplications are unstable, and several revertants of each were sequenced. Most of the revertants had the wild-type nucleotide sequence; however, one had imprecisely removed the duplicated residues, leaving four new nucleotides compared to the wild-type sequence. These mutations represent significant genetic events with regard to their high rates and their gross structural alterations. As to their origin, the mutations can be described as the end-products of endonuclease cleavage of DNA at regions of potential secondary structure and subsequent DNA synthesis. The secondary structure contains four base-paired stems that emerge from duplex DNA. These stems encode the anticodon and variable arm regions of the tRNA(Ser) molecule. The cleavage sites mimic the known substrate of T4 endonuclease VII, an enzyme previously noted for its ability to resolve Holliday-like DNA intermediates.

Rules that govern tRNA identity in protein synthesis.

The specificity of tRNA in protein synthesis depends not only on its recognition of the codon in the mRNA, but also on its recognition of the correct aminoacyl-tRNA synthetase enzyme. The specificity of tRNA in aminoacylation (tRNA identity) depends on the tRNAs productive interaction with the correct enzyme and non-productive interaction with all other enzymes. Although extensive regions of the tRNA interact with the enzyme, only a small number of nucleotides comprise the major determinants of tRNA identity. They often lie in the same positions (acceptor end and anticodon, and variable pocket less often) in different tRNAs. Therefore, a determinant in a given tRNA simultaneously ensures both productive and non-productive interactions with the respective enzymes. Specificity for the acceptor end of the tRNA is achieved, in part, by the specific amino acid sequence within protein binding pocket domains that are part of all aminoacyl-tRNA synthetases. These domains also bind the other two substrates of the enzyme, amino acid and ATP. Specificity for the anticodon and variable pocket of the tRNA is more idiosyncratic. Irrespective of their location in the tRNA, the determinants either interact directly with the enzyme or give the tRNA a conformation for a complementary fit with the enzyme.

Genetic conversion of G.C base-pairs to A.U base-pairs in a transfer RNA.

The cloverleaf stem segments of the suppressor gene of bacteriophage T4 tRNA(Gln) contain ten G.C and ten A.U base-pairs. To gain a better appreciation of the G.C base-pair requirement, we isolated multiple mutants of this suppressor gene in which base-pairs of G.C were replaced by A.U. One active suppressor gene contained only A.U base-pairs on the anticodon stem, indicating that G.C base-pairs in this region of tRNA(Gln) are not essential for function. In contrast, replacement was not possible at two base-pairs on the D stem and at one base-pair on the T stem.

Nucleotides that contribute to the identity of Escherichia coli tRNA(Phe).

A series of sequence variants of amber suppressor genes of tRNA(Phe) were synthesized in vitro and cloned in Escherichia coli to examine the contributions of individual nucleotides to identity for amino acid acceptance. Three different but complementary types of tRNA variants were constructed. The first involved the substitution of base-pairs on the cloverleaf stem regions of the E. coli tRNA(Phe). The second type of variant involved total gene synthesis based on wild-type tRNA(Phe) sequences found in Bacillus subtilis and in Halobacterium volcanii. In the third type of variant, the identity of E. coli tRNALys was changed to that of tRNA(Phe). The nucleotides which are important for tRNA(Phe) identity in E. coli are located on the corner of the L-shaped tRNA molecule, where the dihydrouridine loop interacts with the T loop, and extend to the interior opening of the anticodon stem and the adjoining variable loop. The nucleotide sequence on the dihydrouridine stem region, which joins the corner and stem regions, was not successfully studied though it may contribute to tRNA(Phe) identity. The fourth nucleotide from the 3' end of tRNA(Phe) has some importance for identity.

Construction of an Escherichia coli knockout strain for functional analysis of tRNA(Asp).

The specific aminoacylation of tRNA is critical for translation of the genetic code. A molecular description of aminoacylation requires knowledge of the relevant three-dimensional structures, biochemical parameters and the structure-function relationship of the synthetase and its substrate tRNA. Extensive structural and biochemical data are available on the aspartic acid system of Escherichia coli, but there is a paucity of cellular functional data. We have developed a system to overcome this deficiency by engineering an E. coli knockout tRNA(Asp) strain, thereby allowing a penetrating analysis of tRNA(Asp) structure and function under conditions that prevail in the cell.

Plasmid systems to study RNA function in Escherichia coli.

Determining the functional activity of an essential RNA in vivo presents special challenges. We have devised an in vivo analysis of alternative forms of an essential tRNA gene in Escherichia coli knockout cells using either a plasmid switch or a regulated two-plasmid system. The model system is presented together with a description of the new plasmids and procedures necessary to effect these analyses. The system is readily adaptable to non-essential RNAs.

Differences between transfer RNA molecules.

Computer-assisted comparisons of 67 tRNA sequences that function in Escherichia coli or Salmonella typhimurium were used to identify single and multiple nucleotide positions that maximally distinguish the 20 amino acid acceptor groups. Positions in the anticodon were identified most frequently, as expected from the decoding function of this region of the tRNA. The biological function, if any, of positions outside the anticodon may include specificity for aminoacyl-tRNA synthetase enzymes.

A set of plasmids constitutively producing different RNA levels in Escherichia coli.

New plasmids were developed for the in vivo expression of RNA in Escherichia coli. These plasmids combine constitutive promoters of different strengths with different origins of replication to provide a 75-fold range of expression of amber suppressor tRNA. The plasmids are either pMB1, p15A or temperature-sensitive SC101 replicons, and can be used in two plasmid systems for studying RNA-protein interactions. The temperature-sensitive SC101 plasmids may be useful as gene replacement vectors. Another vector that is suitable for generating lethal mutations was constructed in a plasmid containing a regulatable phage T7 promoter.

Genetic analysis of structure and function in phage T4 tRNASer.

We have determined the nucleotide sequences of 55 spontaneous mutations that inactivate a suppressor gene of phage T4 tRNASer. Most of the mutations caused substitutions or deletions of single nucleotides at 18 different positions in the tRNA. Two of three mutations that allowed the synthesis of mature tRNA had nucleotide substitutions at the junction of the dihydrouridine and anticodon stems, suggesting that this region of tRNASer is important for aminoacylation. The third mutation that synthesized tRNA had a nucleotide deletion in the anticodon loop, which presumably affected the translational capacity of the tRNA. We also sequenced 58 spontaneous reversion mutations derived from strains with the inactive suppressor genes. Some of these regenerated the initial tRNA sequence, while other generated a second-site mutation in the tRNA. These second-site mutations restored helical base-pairings to the tRNA that had been eliminated by the initial mutations. The new base-pairings involved G.C and A.U, and the A.C wobble pair at certain positions in the tRNA. This finding establishes the existence of A.C wobble pair in tRNA helices.

Suppressor and novel mutants of bacteriophage T4 tRNA(Gly).

We have isolated a weak UGA suppressor of phage T4 tRNA(Gly) in which the anticodon is changed from UCC to UCA. Two secondary mutants lacking suppressor activity are atypical in accumulating tRNA(Gly). Both mutations change the T stem of the cloverleaf model. One involved a G to A change at the 5' base position of the middle base-pair; the second involves a C to U change at a constant base position next to the T loop. The precursor RNAs of the mutants were cleaved in vitro with the catalytic RNA subunit of RNase P. Relative to normal precursor RNA, the precursor mutated at the middle base-pair position of the T stem was cleaved more rapidly, whereas the precursor mutated at the base-pair position next to the T loop was cleaved more slowly.

Functional compensation by particular nucleotide substitutions of a critical G*U wobble base-pair during aminoacylation of transfer RNA.

Expression of the genetic code depends on precise tRNA aminoacylation by cognate aminoacyl-tRNA synthetase enzymes. The G.U wobble base-pair in the acceptor helix of Escherichia coli alanine tRNA is the primary aminoacylation determinant of this molecule. Previous work on the process of synthetase recognition of the G.U pair showed that replacing G.U by a G.C Watson-Crick base-pair inactivates alanine acceptance by the tRNA, but that C.A and G.A wobble pair replacements preserve acceptance. Work by another group reported that the effects of a G.C replacement were reversed by a distal wobble base-pair in the anticodon helix. This result is potentially interesting because it suggests that distant regions in alanine tRNA are functionally coupled during synthetase recognition and more generally because recognition determinants of many other tRNAs lie in both the acceptor helix and anticodon helix region. Here, we have conducted an extensive in vivo analysis of the distal wobble pair in alanine tRNA and report that it does not behave like a compensating mutation. Restoration of alanine acceptance was not detected even when the synthetase enzyme was overproduced. We discuss the previous experimental evidence and suggest how the distal wobble pair was incorrectly analyzed. The available data indicate that all principal recognition determinants of alanine tRNA lie in the molecule's acceptor helix.

The reliability of in vivo structure-function analysis of tRNA aminoacylation.

The G.U wobble base-pair in the acceptor helix of Escherichia coli tRNAAlais critical for aminoacylation by the alanine synthetase. Previous work by several groups probed the mechanism of enzyme recognition of G.U by a structure-function analysis of mutant tRNAs using either a cell assay (amber suppressor tRNA) or a test tube assay (phage T7 tRNA substrate and purified enzyme). However, the aminoacylation capacity of particular mutant tRNAs was about 10(4)-fold higher in the cell assay. This led us to scrutinize the cell assay to determine if any parameter exaggerates the extent of aminoacylation in mutants forming substantial amounts of alanyl-tRNAAla. In doing so, we have refined and developed experimental designs to analyze tRNA function. We examined the level of aminoacylation of amber suppressor tRNAAlawith respect to the method of isolating aminoacyl-tRNA, the rate of cell growth, the cellular levels of alanine synthetase and elongation factor TU (EF-Tu), the amount of tRNA and the characteristics of EF-Tu binding. Within the precision of our measurements, none of these parameters varied in a way that could significantly amplify cellular alanyl-tRNAAla. A key observation is that the extent of aminoacylation of tRNAAlawas independent of tRNAAlaconcentration over a 75-fold range. Therefore, the cellular assay of tRNAAlareflects the substrate quality of the molecule for formation of alanyl-tRNAAla. These experiments support the authenticity of the cellular assay and imply that a condition or factor present in the cell assay may be absent in the test tube assay.

Rapid site-specific mutagenesis in plasmids.

A quick and simple method for introducing site-specific mutations into plasmids is described. The procedure involves restriction-enzyme digestion of the plasmid to give a linear fragment. A second preparation of the same plasmid is digested with other restriction enzymes to remove the targeted mutational region to give a gapped fragment. The linear fragment and the gapped fragment are mixed, then denatured and annealed in the presence of a short, synthetic oligodeoxynucleotide corresponding to the targeted region and containing the desired mutation. The mix is then transformed directly into cells where host enzymes fill single-stranded gaps to make a complete double-stranded, mutant plasmid.

Association of tRNA(Gln) acceptor identity with phosphate-sugar backbone interactions observed in the crystal structure of the Escherichia coli glutaminyl-tRNA synthetase-tRNA(Gln) complex.

We isolated several mutants with nucleotide substitutions in alanine tRNA (tRNA(Ala)) that resulted in glutamine tRNA (tRNA(Gln)) acceptor identity in Escherichia coli. These substitutions were in three regions of tRNA structure not previously associated with tRNA(Gln) acceptor identity. Only the phosphate-sugar backbone moieties of these nucleotides interact with the enzyme in the previously determined X-ray crystal structure of the complex between tRNA(Gln) and glutaminyl-tRNA synthetase. We conclude that these sequence-dependent phosphate-sugar backbone interactions contribute to tRNA(Gln) identity, and argue that the interactions help communicate enzyme recognition of the anticodon to the acceptor end of the tRNA and the catalytic center of the enzyme.

Isolation of novel tRNA(Ala) mutants by library selection in a tRNA(Ala) knockout strain.

The relationship between tRNA structure and function has been widely investigated by site-directed mutagenesis. This method has been a very useful tool to reveal the critical bases in tRNAs that are important for recognition and aminoacylation, but has been limited by the large number of possible base combinations in tRNA molecules. We have devised a new method that uses tRNA knockout cells for selection of functional tRNAs from a mutant tRNA gene library to overcome this limitation. To explore the mechanism of tRNA(Ala) recognition, the bases of the acceptor-stem region were randomized and active mutants were selected in a tRNA(Ala) knockout strain. Mutants of tRNA(Ala) having diverse sequence combinations in the acceptor-stem region and a broad range of functional activity to support knockout cell growth were isolated. The mutant tRNAs selected by the method included molecules containing novel base substitutions as well as extensively altered base combinations that would not be readily generated by rationally designed site-directed mutagenesis. Our results emphasize the importance of the acceptor stem as a structural unit in which some nucleotides may carry more weight than others, but in summation every nucleotide contributes to the interaction with the enzyme.