Studies on anticodon-anticodon interactions: hemi-protonation of cytosines induces self-pairing through the GCC anticodon of E. coli tRNA-Gly

The temperature-jump method was used to compare the stability of anticodon-anticodon duplexes formed by the self-association of two tRNAs: yeast tRNA-Asp and Escherichia coli tRNA-Gly. Yeast tRNA-Asp duplexes contain a U/U mismatch while E. coli tRNA-Gly dimers have a C/C mismatch in the middle position of their quasi self-complementary anticodons GUC and GCC, respectively. At neutral pH, it is found that only tRNA-Asp duplexes exist whereas at pH 5.0 only tRNA-Gly duplexes are formed. This reflects the hemiprotonation of the N3 of the cytosines at pH 5.0 which induces a pairing between the two middle residues of the anticodon GCC in E. coli tRNA-Gly. This is the first evidence that a protonated C-C(+) base pair is compatible with the formation of a double helix with antiparallel strands in a natural RNA molecule.     

Enzymatic formation of queuosine and of glycosyl queuosine in yeast tRNAs microinjected into Xenopus laevis oocytes. The effect of the anticodon loop sequence

The eukaryotic tRNA-guanine transglycosylases (queuine insertases) catalyse an exchange of guanine for queuine in position 34, the wobble nucleoside, of tRNAs having a GUN anticodon where N (position 36) stands for A, U, C or G. In tRNAAsp (anticodon QUC) and tRNATyr (anticodon Q psi A) from certain eukaryotic cells, the nucleoside Q-34 is further hypermodified into a glycosylated derivative by tRNA-queuine glycosyltransferase. In order to gain insight into the influence of the nucleosides in position 36, 37 and 38 of an anticodon loop on the potential of a tRNA to become a substrate for the two modifying enzymes, we have constructed several variants of yeast tRNAs in which the normal anticodon has been replaced by one of the synthetic anticodons GUA, GUC, GUG or GUU. In yeast tRNAAsp, the nucleosides 37 (m1G) and 38(C) have also been replaced by an adenosine. These reconstructed chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and tested for their ability to react with the oocyte maturation enzymes. Our results indicate that the nucleosides in positions 36, 37 and 38 influence the efficiencies of conversion of G-34 to Q-34 and of Q-34 to glycosyl Q-34; the importance of their effects are much more pronounced on the glycosylation of Q-34 than on the insertion of queuine. The effect of the nucleoside in position 37 is of particular importance in the case of yeast tRNAAsp: the replacement of the naturally occurring m1G-37 by an unmodified adenosine (as it is in X. laevis tRNAAsp), considerably increases the yield of the glycosylation reaction catalysed by the X. laevis tRNA-queuine glycosyltransferase.     

Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence.

We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs. For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U). This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34. Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme. Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all. This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.     

Studies on Anticodon-anticodon Interactions: Hemi-protonation of Cytosines Induces Self-pairing Through the GCC Anticodon of E. Coli tRNA-Gly

Abstract

The temperature-jump method was used to compare the stability of anticodon-anticodon duplexes formed by the self-association of two tRNAs: yeast tRNA-Asp and Escherichia coli tRNA-Gly. Yeast tRNA-Asp duplexes contain a U/U mismatch while E. coli tRNA-Gly dimers have a C/C mismatch in the middle position of their quasi self-complementary anticodons GUC and GCC, respectively. At neutral pH, it is found that only tRNA-Asp duplexes exist whereas at pH 5.0 only tRNA-Gly duplexes are formed. This reflects the hemi- protonation of the N3 of the cytosines at pH 5.0 which induces a pairing between the two middle residues of the anticodon GCC in E. coli tRNA-Gly. This is the first evidence that a protonated C-C(+) base pair is compatible with the formation of a double helix with antiparallel strands in a natural RNA molecule.     

Fluorine-19 nuclear magnetic resonance study of codon-anticodon interaction in 5-fluorouracil-substituted E. coli transfer RNAs.

Codon-anticodon interaction was investigated in fully active 5-fluorouracil-substituted E. coli tRNAVal1 (anticodon FAC) by 19F NMR spectroscopy. Binding of the codon GpUpA results in the upfield shift of a 19F resonance at 3.9 ppm in the central region of the 19F NMR spectrum, whereas trinucleotides not complementary to the anticodon have no effect. The same 19F resonance shifts upfield upon formation of an anticodon-anticodon dimer between the 19F-labeled tRNA and E. coli tRNATyr2 (anticodon QUA). These results permit assignment of the peak at 3.9 ppm to the 5-fluorouracil at position 34 in the anticodon of fluorouracil-substituted tRNAVal1. The methionine codon ApUpG also causes a sequence-specific upfield shift of a peak in the central part of the 19F NMR spectrum of fluorinated E. coli tRNAMetm. However, ApUpG has no effect on the 19F spectrum of 19F-labeled E. coli tRNAMetf, indicating possible conformational differences between the anticodon loop of initiator and chain-elongating methionine tRNAs. 19F NMR experiments detect no binding of CpGpApA to the complementary FpFpCpG (replaces Tp psi pCpG) in the T-loop of 5-fluorouracil-substituted tRNAVal1, in the presence or absence of codon, suggesting that the tertiary interactions between the T- and D-loops are not disrupted by codon-anticodon interactions.     

An anticodon change switches the identity of E. coli tRNA(mMet) from methionine to threonine.

Recent evidence indicates that the anticodon may often play a crucial role in selection of tRNAs by aminoacyl-tRNA synthetases. In order to quantitate the contribution of the anticodon to discrimination between cognate and noncognate tRNAs by E. coli threonyl-tRNA synthetase, derivatives of the E. coli elongator methionine tRNA (tRNA(mMet)) containing wild type and threonine anticodons have been synthesized in vitro and assayed for threonine acceptor activity. Substitution of the threonine anticodon GGU for the methionine anticodon CAU increased the threonine acceptor activity of tRNA(mMet) by four orders of magnitude while reducing methionine acceptor activity by an even greater amount. These results indicate that the anticodon is the major element which determines the identity of both threonine and methionine tRNAs.     

The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs

We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: the stability of these eubacterial tRNA variants in the eukaryotic oocytes; the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-beta-D-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.     

Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of mutants of 5-fluorouracil-substituted Escherichia coli valine tRNA.

In order to utilize 19F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNAVal labeled by incorporation of 5-fluorouracil, we have assigned its 19F spectrum. We describe here assignments made by examining the spectra of a series of tRNAVal mutants with nucleotide substitutions for individual 5-fluorouracil residues. The result of base replacements on the structure and function of the tRNA are also characterized. Mutants were prepared by oligonucleotide-directed mutagenesis of a cloned tRNAVal gene, and the tRNAs transcribed in vitro by bacteriophage T7 RNA polymerase. By identifying the missing peak in the 19F NMR spectrum of each tRNA variant we were able to assign resonances from fluorouracil residues in loop and stem regions of the tRNA. As a result of the assignment of FU33, FU34 and FU29, temperature-dependent spectral shifts could be attributed to changes in anticodon loop and stem conformation. Observation of a magnesium ion-dependent splitting of the resonance assigned to FU64 suggested that the T-arm of tRNAVal can exist in two conformations in slow exchange on the NMR time scale. Replacement of most 5-fluorouracil residues in loops and stems had little effect on the structure of tRNAVal; few shifts in the 19F NMR spectrum of the mutant tRNAs were noted. However, replacing the FU29.A41 base-pair in the anticodon stem with C29.G41 induced conformational changes in the anticodon loop as well as in the P-10 loop. Effects of nucleotide substitution on aminoacylation were determined by comparing the Vmax and Km values of tRNAVal mutants with those of the wild-type tRNA. Nucleotide substitution at the 3' end of the anticodon (position 36) reduced the aminoacylation efficiency (Vmax/Km) of tRNAVal by three orders of magnitude. Base replacement at the 5' end of the anticodon (position 34) had only a small negative effect on the aminoacylation efficiency. Substitution of the FU29.A41 base-pair increased the Km value 20-fold, while Vmax remained almost unchanged. The FU4.A69 base-pair in the acceptor stem, could readily be replaced with little effect on the aminoacylation efficiency of E. coli tRNAVal, indicating that this base-pair is not an identity element of the tRNA, as suggested by others.     

Temperature jump relaxation studies on the interactions between transfer RNAs with complementary anticodons. The effect of modified bases adjacent to the anticodon triplet

We have used the temperature-jump relaxation technique to determine the kinetic and thermodynamic parameters for the association between the following tRNAs pairs having complementary anticodons: tRNA(Ser) with tRNA(Gly), tRNA(Cys) with tRNA(Ala) and tRNA(Trp) with tRNA(Pro). The anticodon sequence of E. coli tRNA(Ser), GGA, is complementary to the U*CC anticodon of E. coli tRNA(Gly(2] (where U* is a still unknown modified uridine base) and A37 is not modified in none of these two tRNAs. E. coli tRNA(Ala) has a VGC anticodon (V is 5-oxyacetic acid uridine) while tRNA(Cys) has the complementary GCA anticodon with a modified adenine on the 3' side, namely 2-methylthio N6-isopentenyl adenine (mS2i6A37) in E. Coli tRNA(Cys) and N6-isopentenyl adenine (i6A37) in yeast tRNA(Cys). The brewer yeast tRNA(Trp) (anticodon CmCA) differs from the wild type E. coli tRNA(Trp) (anticodon CCA) in several positions of the nucleotide sequence. Nevertheless, in the anticodon loop, only two interesting differences are present: A37 is not modified while C34 at the first anticodon position is modified into a ribose 2'-O methyl derivative (Cm). The corresponding complementary tRNA is E.coli tRNA(Pro) with the VGG anticodon. Our results indicate a dominant effect of the nature and sequence of the anticodon bases and their nearest neighbor in the anticodon loop (particularly at position 37 on the 3' side); no detectable influence of modifications in the other tRNA stems has been detected. We found a strong stabilizing effect of the methylthio group on i6A37 as compared to isopentenyl modification of the same residue. We have not been able so far to assess the effect of isopentenyl modification alone in comparison to unmodified A37. The results obtained with the complex yeast tRNA(Trp)-E.coli tRNA(Pro) also suggest that a modification of C34 to Cm34 does not significantly increase the stability of tRNA(Trp) association with its complementary anticodon in tRNA(Pro). The observations are discussed in the light of inter- and intra-strand stacking interactions among the anticodon triplets and with the purine base adjacent to them, and of possible biological implications.     

Site-directed in vitro replacement of nucleosides in the anticodon loop of tRNA: application to the study of structural requirements for queuine insertase activity.

We have investigated the specificity of the enzymes Q-insertase and mannosyl-Q transferase that replace the guanosine at position 34 (wobble base) in the anticodon of several tRNAs by Q or mannosyl-Q derivatives. We have restructured in vitro the normal anticodon of yeast tRNA-Asp-GUC, yeast tRNAArgICG and yeast tRNALeuUAG. With yeast tRNA-Asp-GUC, we have replaced one or several nucleotides in the vicinity of G34 by one of the four canonical nucleotides or by pseudouridylic acid; we have also constructed a tRNAAsp with eight bases instead of seven in the anticodon loop. With yeast tRNAArgICG and yeast tRNALeuUAG, we have replaced their anticodon by the trinucleotide GUC, coding for aspartic acid. The chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and after 72 h the amount of Q34 and mannosyl-Q34 incorporated was measured. Our results show that the U33G34U35 sequence, within an anticodon loop of seven bases in chimerical yeast tRNA-Asp-GUC, tRNAArgGUC or tRNALeuGUC, is the main determinant for Q-insertase activity at position 34; the rest of the tRNA sequence has only a slight influence. For mannosyl-Q transferase, however, a much broader structural feature of the tRNA than just the U33G34U35 sequence is important for the efficiency of Q34 transformation into mannosyl-Q34.     

On the biosynthesis of 5-methoxyuridine and uridine-5-oxyacetic acid in specific procaryotic transfer RNAs

The uridine-5-0-derivatives, 5-methoxyuridine (mo5U) and uridine-5-oxyacetic acid (cmo5U) occupy the first position of anticondons in certain tRNA species of B. subtilis and E. coli, respectively. Here we present experimental evidence showing that both modifications are derived from a common precursor, 5-hydroxyuridine. Incompletely modified tRNASer and tRNAVal from E. coli met- rel-. All five tRNAs accepted methyl groups from S-adenosylmethionine with B. subtilis extracts in vitro and mo5U was formed. In B. subtilis tRNAs the mo5U was proved to be at the specific site; in E. coli tRNAVal the mo5U was demonstrated to be present in the oligonucleotide that comprises the anticodon. In submethylated E. coli tRNAVal,5-hydroxyuridine was detected whereas considerable amounts of cmo5U were lacking.     

Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli

Naturally occurring tRNA mutants are known that suppress +1 frameshift mutations by means of an extended anticodon loop, and a few have been used in protein mutagenesis. In an effort to expand the number of possible ways to uniquely and efficiently encode unnatural amino acids, we have devised a general strategy to select tRNAs with the ability to suppress four-base codons from a library of tRNAs with randomized 8 or 9 nt anticodon loops. Our selectants included both known and novel suppressible four-base codons and resulted in a set of very efficient, non-cross-reactive tRNA/four-base codon pairs for AGGA, UAGA, CCCU and CUAG. The most efficient four-base codon suppressors had Watson-Crick complementary anticodons, and the sequences of the anticodon loops outside of the anticodons varied with the anticodon. Additionally, four-base codon reporter libraries were used to identify "shifty" sites at which +1 frameshifting is most favorable in the absence of suppressor tRNAs in Escherichia coli. We intend to use these tRNAs to explore the limits of unnatural polypeptide biosynthesis, both in vitro and eventually in vivo. In addition, this selection strategy is being extended to identify novel five- and six-base codon suppressors.     

The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression

tRNA species in Escherichia coli that translate codons starting with U contain 2-methyl-thio-N6-isopentenyl-adenosine in position 37, 3' adjacent to the anticodon. The role of this hypermodification in protein synthesis and trp operon attenuation has been investigated. Temperature-jump relaxation methods have been applied to study the interaction between E. coli tRNAPro, with anticodon VGG (V is uridine-5-oxyacetic acid) complementary to that of tRNATrp, and three species of E. coli tRNATrp: wild type tRNATrp (with ms2i6A37 and G24), UGA suppressor tRNATrp (with ms2i6A37 and A24 in the dihydrouridine stem but the same anticodon CCA), and the same suppressor molecule but ms2i6A-deficient as a result of the mutation miaA. Complex formation between tRNAPro and ms2i6A-containing tRNATrp shows thermodynamic parameters close to those found for several other pairs of tRNA with complementary anticodons. However, ms2i6A-deficient tRNATrp makes less stable complexes with tRNAPro, which dissociate eightfold faster. No effect on the complementary anticodon interaction of the mutation in the dihydrouridine stem can be detected. When the tRNA analogous to the opal codon, E. coli tRNASerIV (anticodon VGA) replaces tRNAPro in similar experiments, very weak complexes are observed with both normally hypermodified species of tRNATrp, the wild type and UGA suppressor; these show a lifetime about 50-fold shorter than with tRNAPro, but are again similar. No complex formation is detectable with the ms2i6A-deficient species. This may explain why the hypermodification is necessary for the efficient suppression of the UGA terminator of Q beta coat protein in vitro. The data on complexes with tRNAPro suggest that deficiency in ms2i6A may also reduce the efficiency of UGG reading. Thus, miaA may affect trp operon attenuation by slowing translation of the tandem UGG codons in the leader sequence. Temperature-jump differential spectra suggest that ms2i6 stabilizes the anticodon interaction by improved stacking of base 37.     

The primary structure of tRNA Val 2a from baker's yeast]

The primary structure of tRNAVal2a from baker's yeast has been determined. The general methods of the investigation are presented. Twenty six distinguished points can be noted in the tRNAVal2a and tRNA1Val from baker's yeast. The anticodon region of tRNAVal2a is represented by the sequence NAC, where N corresponds to a uridine analogue nucleoside of unknown structure. The comparison of primary structures of tRNAVal2a, tRNAVal2a, tRNA1Val from E. coli and tRNAVal2a and tRNA1Val from baker's yeast is analysed.     

The influence of elongation-factor-Tu . GTP and anticodon-anticodon interactions on the anticodon loop conformation of yeast tRNATyr

The interactions of yeast tRNATyr, spin-labelled at position i6A-37 next to the anticodon, with EF-Tu . GTP and with Escherichia coli tRNAVal (which has a complementary anticodon) have been studied. The immobilization of the spin label upon ternary complex formation shows a conformational change of the anticodon region, although this part of tRNATyr is not in direct contact with the protein, as indicated by RNase T1 digestion. Upon anticodon-anticodon interaction, no conformational change of the anticodon loop of tRNATyr was observed.     

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study.

Interactions of 5-fluorouracil-substituted Escherichia coli tRNAVal with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene [Heck, J.D., & Hatfield, G.W. (1988) J. Biol. Chem. 263, 868-877]. Apparent KM and Vmax values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNAVal. Binding of VRS to (FUra)tRNAVal induces structural perturbations that are reflected in selective changes in the 19F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNAVal along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNAVal, suggesting conformational changes in this part of the molecule. No 19F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNAVal that has been proposed as a common intermediate in the aminoacylation reaction.     

Imino proton NMR assignments and ion-binding studies on Escherichia coli tRNA3Gly

The imino region of the proton NMR spectrum of Escherichia coli tRNA3Gly has been assigned mainly by sequential nuclear Overhauser effects between neighbouring base pairs and by comparison of assignments of other tRNAs. The effects of magnesium, spermine and temperature on the 1H and 31P NMR spectra of this tRNA were studied. Both ions affect resonances close to the G15 . C48 tertiary base pair and in the ribosylthymine loop. The magnesium studies indicate the presence of an altered tRNA conformer at low magnesium concentrations in equilibrium with the high magnesium form. The temperature studies show that the A7 . U66 imino proton (from a secondary base pair) melts before some of the tertiary hydrogen bonds and that the anticodon stem does not melt sequentially from the ends. Correlation of the ion effects in the 1H and 31P NMR spectra has led to the tentative assignment of two 31P resonances not assigned in the comparable 31P NMR spectrum of yeast tRNAPhe. 31P NMR spectra of E. coli tRNA3Gly lack resolved peaks corresponding to peaks C and F in the spectra of E. coli tRNAPhe and yeast tRNAPhe. In the latter tRNAs these peaks have been assigned to phosphate groups in the anticodon loop. Ion binding E. coli tRNA3Gly and E. coli tRNAPhe had different effects on their 1H NMR spectra which may reflect further differences in their charge distribution and conformation.     

Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl-tRNA synthetases,methionyl-tRNA formyltransferase,and initiation factor 2 on activity in initiation

Anticodon sequence mutants of Escherichia coli initiator tRNA initiate protein synthesis with codons other than AUG and amino acids other than methionine. Because the anticodon sequence is, in many cases, important for recognition of tRNAs by aminoacyl-tRNA synthetases, the mutant tRNAs are aminoacylated in vivo with different amino acids. The activity of a mutant tRNA in initiation in vivo depends on (i) the level of expression of the tRNA, (ii) the extent of aminoacylation of the tRNA, (iii) the extent of formylation of the aminoacyl-tRNA to formylaminoacyl-tRNA (fAA-tRNA), and (iv) the affinity of the fAA-tRNA for the initiation factor IF2 and the ribosome. Previously, using E. coli overproducing aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, or IF2, we identified the steps limiting the activity in initiation of mutant tRNAs aminoacylated with glutamine and valine. Here, we have identified the steps limiting the activity of mutant tRNAs aminoacylated with isoleucine and phenylalanine. The combined results of experiments involving a variety of initiation codons (AUG, UAG, CAG, GUC, AUC, and UUC) provide support to the hypothesis that the ribosome.fAA-tRNA complex can act as an intermediate in initiation of protein synthesis. Comparison of binding affinities of various fAA-tRNAs (fMet-, fGln-, fVal-, fIle-, and fPhe-tRNAs) to IF2 using surface plasmon resonance supports the idea that IF2 can act as a carrier of fAA-tRNA to the ribosome. Other results suggest that the C1xA72 base pair mismatch, unique to eubacterial and organellar initiator tRNAs, may also be important for the binding of fAA-tRNA to IF2.