Acceptor stem and anticodon RNA hairpin helix interactions with glutamine tRNA synthetase

The class I glutamine (Gln) tRNA synthetase interacts with the anticodon and acceptor stem of glutamine tRNA. RNA hairpin helices were designed to probe acceptor stem and anticodon stem-loop contacts. A seven-base pair RNA microhelix derived from the acceptor stem of tRNA^Gln was aminoacylated by Gln tRNA synthetase. Variants of the glutamine acceptor stem microhelix implicated the discriminator base as a major identity element for glutaminylation of the RNA helix. A second RNA microhelix representing the anticodon stem-loop competitively inhibited tRNA^Gln charging. However, the anticodon stem-loop microhelix did not enhance aminoacylation of the acceptor stem microhelix. Thus, transduction of the anticodon identity signal may require covalent continuity of the tRNA chain to trigger efficient aminoacylation.     

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem

We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A. "mismatch" in E. coli initiator tRNA. Mutation of this C.A. to U:A, a weak base pair, or U.G., a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kappm by 495-fold. Mutation of the second basepair G2:C71 to U2:A71 lowers Vmax/Kappm by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kappm by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and T psi C stem have little or no effect on formylation.     

Role of acceptor stem conformation in tRNAVal recognition by its cognate synthetase.

Although the anticodon is the primary element in Escherichia coli tRNAValfor recognition by valyl-tRNA synthetase (ValRS), nucleotides in the acceptor stem and other parts of the tRNA modulate recognition. Study of the steady state aminoacylation kinetics of acceptor stem mutants of E.coli tRNAValdemonstrates that replacing any base pair in the acceptor helix with another Watson-Crick base pair has little effect on aminoacylation efficiency. The absence of essential recognition nucleotides in the acceptor helix was confirmed by converting E.coli tRNAAlaand yeast tRNAPhe, whose acceptor stem sequences differ significantly from that of tRNAVal, to efficient valine acceptors. This transformation requires, in addition to a valine anticodon, replacement of the G:U base pair in the acceptor stem of these tRNAs. Mutational analysis of tRNAValverifies that G:U base pairs in the acceptor helix act as negative determinants of synthetase recognition. Insertion of G:U in place of the conserved U4:A69 in tRNAValreduces the efficiency of aminoacylation, due largely to an increase in K m. A smaller but significant decline in aminoacylation efficiency occurs when G:U is located at position 3:70; lesser effects are observed for G:U at other positions in the acceptor helix. The negative effects of G:U base pairs are strongly correlated with changes in helix structure in the vicinity of position 4:69 as monitored by19F NMR spectroscopy of 5-fluorouracil-substituted tRNAVal. This suggests that maintaining regular A-type RNA helix geometry in the acceptor stem is important for proper recognition of tRNAValby valyl-tRNA synthetase.19F NMR also shows that formation of the tRNAVal-valyl-tRNA synthetase complex does not disrupt the first base pair in the acceptor stem, a result different from that reported for the tRNAGln-glutaminyl-tRNA synthetase complex.     

Role of the three consecutive G:C base pairs conserved in the anticodon stem of initiator tRNAs in initiation of protein synthesis in Escherichia coli.

The three consecutive G:C base pairs, G29:C41, G30:C40, and G31:C39, are conserved in the anticodon stem of virtually all initiator tRNAs from eubacteria, eukaryotes, and archaebacteria. We show that these G:C base pairs are important for function of the tRNA in initiation of protein synthesis in vivo. We changed these base pairs individually and in combinations and analyzed the activities of the mutant Escherichia coli initiator tRNAs in initiation in vivo. For assessment of activity of the mutant tRNAs in vivo, mutations in the G:C base pairs were coupled to mutation in the anticodon sequence from CAU to CUA. Mutations in each of the G:C base pairs reduced activity of the mutant tRNA in initiation, with mutation in the second G:C base pair having the most severe effect. The greatly reduced activity of this C30:G40 mutant tRNA is not due to defects in aminoacylation or formulation of the tRNA or defects in base modification of the A37, next to the anticodon, which we had previously shown to be important for activity of the mutant tRNAs in initiation. The anticodon stem mutants are most likely affected specifically at the step of binding to the ribosomal P site. The pattern of cleavages in the anticodon loop of mutant tRNAs by S1 nuclease indicate that the G:C base pairs may be involved directly in interactions of the tRNA with components of the P site on the ribosome rather than indirectly by inducing a particular conformation of the anticodon loop critical for function of the tRNA in initiation.     

The exchange of the discriminator base A73 for G is alone sufficient to convert human tRNA(Leu) into a serine-acceptor in vitro.

Transfer RNA (tRNA) identify is maintained by the highly specific interaction of a few defined nucleotides or groups of nucleotides, called identity elements, with the cognate aminoacyl-tRNA synthetase, and by nonproductive interactions with the other 19 aminoacyl-tRNA synthetases. Most tRNAs have a set of identity elements in at least two locations, commonly in the anticodon loop or in the acceptor stem, and at the discriminator base position 73. We have used T7 RNA polymerase transcribed tRNAs to demonstrate that the sole replacement of the discriminator base A73 of human tRNA(Leu) with the tRNA(Ser)-specific G generates a complete identity switch to serine acceptance. The reverse experiment, the exchange of G73 in human tRNA(Ser) for the tRNA(Leu-specific A, causes a total loss of serine specificity without creating any leucine acceptance. These results suggest that the discriminator base A73 of human tRNA(Leu) alone protects this tRNA against serylation by seryl-tRNA synthetase. This is the first report of a complete identity switch caused by an exchange of the discriminator base alone.     

Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.     

Minimum requirements for substrates of mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes a 3' trailer after the discriminator nucleotide from precursor tRNA (pre-tRNA). To elucidate the minimum requirements for 3' tRNase substrates, we tested small pre-tRNA(Arg) substrates lacking the D and anticodon stem-loop domain for cleavage by purified pig 3' tRNase. A small pre-tRNA (R-ATW) composed of an acceptor stem, an extra loop, a T stem-loop domain, a discriminator nucleotide, and a 3' trailer was cleaved more efficiently than the full-length wild type. The catalytic efficiencies of three R-ATW derivatives, which were constructed to destroy the original T stem base pairs, were also higher than that of the full-length wild type. Pig 3' tRNase efficiently processed a "minihelix" (R-ATM5) that consists of a T stem-loop domain, an acceptor stem, a discriminator nucleotide, and a 3' trailer, while the enzyme never cleaved a "microhelix" that is composed of a T loop, an acceptor stem, a discriminator nucleotide, and a 3' trailer. Five R-ATM5 derivatives that have one to seven base substitutions in the T loop were all cleaved slightly more efficiently than the full-length wild type and slightly less efficiently than R-ATM5. A helix ("minihelixDelta1") one base pair smaller than minihelices was a good substrate, while small helices containing a continuous 10-base pair stem were poor substrates. The cleavage of these three small substrates occurred after the discriminator and one to three nucleotides downstream of the discriminator. From these results, we conclude that minimum substrates for efficient cleavage by mammalian 3' tRNase are minihelices or minihelicesDelta1, in which there seem to be no essential bases.     

Recognition of pyrrolysine tRNA by the Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase

Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the special amber suppressor tRNA(Pyl). The genes for tRNA(Pyl) (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activation and aminoacylation properties of D. hafniense PylRS and the nature of the tRNA(Pyl) identity elements were determined by measuring the ability of 24 mutant tRNA(Pyl) species to be aminoacylated with the pyrrolysine analog N-epsilon-cyclopentyloxycarbonyl-l-lysine. The discriminator base G73 and the first base pair (G1.C72) in the acceptor stem were found to be major identity elements. Footprinting analysis showed that PylRS binds tRNA(Pyl) predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem. Significant contacts with the anticodon arm were not observed. The tRNA(Pyl) structure contains the highly conserved T-loop contact U54.A58 and position 57 is conserved as a purine, but the canonical T- to D-loop contact between positions 18 and 56 was not present. Unlike most tRNAs, the tRNA(Pyl) anticodon was shown not to be important for recognition by bacterial PylRS.