Coli Initiator tRNA Research Articles

Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria.

Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS-suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The "21st synthetase-tRNA pairs" include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold.

Specific N-terminal biotinylation of a protein in vitro by a chemically modified tRNA fmet can support the native activity of the translated protein

Biotinylation of a protein generally involves chemical modification of a translated protein. Using this methodology, however, biotinylation at a specific position remains difficult. We investigated whether it would be possible to use an Escherichia coli initiator tRNA fmet aminoacylated with methionine biotinylated at the α-amino group to introduce a biotin tag specifically at the N terminus. We report here that a biotin tag could be incorporated into the green fluorescent protein (GFP) at the N-terminal site, in the presence of an E. coli initiator tRNA fmet aminoacylated with methionine biotinylated at the α-amino group. The biotinylated GFP was purified by simple monomeric streptavidin-agarose affinity column chromatography. Based on the total amount of GFP molecules, the purification yield and the biotin labelling efficiency of this system were approximately 7% and 10–20%, respectively, according to the densitometric analysis of Western blots. Judging from the results of a fluorescence imaging experiment, almost all the purified GFP molecules retained the native fluorescence activity. Importantly, the present results support the hypothesis that the E. coli initiator tRNA fmet aminoacylated with a relatively large substituent can be recognized by an E. coli ribosome and adequately placed at the P site to initiate translation.

Important Role of the Amino Acid Attached to tRNA in Formylation and in Initiation of Protein Synthesis in Escherichia coli

In attempts to convert an elongator tRNA to an initiator tRNA, we previously generated a mutant elongator methionine tRNA carrying an anticodon sequence change from CAU to CUA along with the two features important for activity of Escherichia coli initiator tRNA in initiation. This mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only when aminoacylated with methionine by overproduction of methionyl-tRNA synthetase. Here we show that the Mi:2 tRNA is normally aminoacylated in vivo with lysine and that the tRNA aminoacylated with lysine is a very poor substrate for formylation compared with the same tRNA aminoacylated with methionine. By introducing further changes at base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in the E. coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tRNA transformylase results in partial formylation of the mutant tRNA and activity of the formyllysyl-tRNAs in initiation of protein synthesis. Thus, the G4: C69 base pair contributes toward formylation of the tRNA and protein synthesis in E. coli can be initiated with formyllysine. We also discuss the implications of these and other results on recognition of tRNAs by E. coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases.

Saccharomyces cerevisiae cytoplasmic tyrosyl-tRNA synthetase gene. Isolation by complementation of a mutant Escherichia coli suppressor tRNA defective in aminoacylation and sequence analysis

Exploiting differences in tRNA recognition between prokaryotic and eukaryotic tyrosyl-tRNA synthetases (TyrRSs), we have isolated the gene for the cytoplasmic TyrRS of Saccharomyces cerevisiae by functional complementation in Escherichia coli of a mutant E. coli tRNA. The tRNA, derived from the E. coli initiator tRNA with changes to allow suppression of amber termination codons, is poorly aminoacylated in E. coli and hence, is only a weak amber suppressor. The same tRNA functions as a good suppressor in S. cerevisiae and is aminoacylated with tyrosine by yeast extracts. We expressed a yeast cDNA library in an E. coli strain carrying the mutant tRNA gene and several genes with amber mutations. cDNA clones were isolated which increased suppression and levels of aminoacylation of the mutant tRNA. Characterization of the gene identified a methionine-initiated open reading frame encoding a protein of 394 amino acids. Expression of this protein in E. coli demonstrated that tyrosine was incorporated during suppression and that yeast cytoplasmic TyrRS activity was produced. Yeast cytoplasmic TyrRS has sequences typical of class I aminoacyl-tRNA synthetases, but only weak overall sequence similarity to the corresponding eubacterial and mitochondrial TyrRSs. However, many of the residues known to line the tyrosyl-adenylate-binding pocket of the Bacillus stearothermophilus enzyme can be aligned in the yeast sequence. These include the aspartic acid and tyrosine residues thought to contact the tyrosine side chain to provide substrate specificity.

Relationship between the structure and function of Escherichia coli initiator tRNA

Through functional studies of mutant tRNAs, we have identified sequence and/or structural features important for specifying the many distinctive properties of E coli initiator tRNA. Many of the mutant tRNAs contain an anticodon sequence change from CAU→CUA and are now substrates for E coli glutaminyl-tRNA synthetase (GlnRS). We describe here the effect of further mutating the discriminator base 73 and nucleotide 72 at the end of the acceptor stem on: i) recognition of the mutant tRNAs by E coli GlnRS; ii) recognition by E coli methionyl-tRNA transformylase; and iii) activity of the mutant tRNAs in initiation in E coli. For GlnRS recognition, our results are, in general, consistent with interactions found in the crystal structure of the E coli GlnRS-glutamine tRNA complex. The results also support our previous conclusion that formylation of initiator tRNA is important for its function in initiation.

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.

The conformation of the initiator tRNA and of the 16S rRNA from Escherichia coli during the formation of the 30S initiation complex

The conformation of the E. coli initiator tRNA and of the 16S rRNA at different steps leading to the 30S · IF2 · fMet-ARNt Met t · AUG · GTP complex has been investigated using several structure-specific probes. As compared to elongator tRNA, the initiator tRNA exhibits specific structural features in the anticodon arm, the T and D loops and the acceptor arm. Initiation factor 2 (IF2) interacts with the T-loop and the minor groove of the T stem of the RNA, and induces an increased flexibility in the anticodon arm. In the 30S initiation complex, additional protection is observed in the acceptor stem and in the anticodon arm of the tRNA. Within the 30S subunit, IF2 does not significantly shield defined portions of 16S rRNA, but induces both reduction and enhancement of reactivity scattered in the entire molecule. Most are constrained in a region corresponding to the cleft, the lateral protrusion and the part of the head facing the protrusion. All the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG message. The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding.

Initiation of protein synthesis from a termination codon.

We show that the amber termination codon UAG can initiate protein synthesis in Escherichia coli. We mutated the initiation codon AUG of the chloramphenicol acetyltransferase (CAT) gene to UAG (CATam1) and translated mRNA derived from the mutant CAT gene in E. coli S-30 extracts. A full-length CAT polypeptide was synthesized in the presence of tRNA(fMetCUA), a mutant E. coli initiator tRNA which has a change in the anticodon sequence from CAU to CUA. Addition of purified E. coli glutaminyl-tRNA synthetase substantially stimulated synthesis of the CAT polypeptide. Thus, initiation of protein synthesis with UAG and tRNA(fMetCUA) most likely occurs with glutamine and not methionine. The UAG codon also initiates protein synthesis in vivo. To eliminate a weak secondary site of initiation from AUC, the fifth codon, we further mutagenized the CATam1 gene at codons 2 (GAG----GAC) and 5 (AUC----ACC). Transformation of E. coli with the resultant CATam1.2.5 gene yielded transformants that synthesized CAT polypeptide and were resistant to chloramphenicol only when they were also transformed with the mutant tRNA(fMetCUA) gene. Immunoblot analyses and assays for CAT enzyme activity in extracts from transformed cells indicate that initiation from UAG is efficient, 60-70% of that obtained from AUG. Initiation of protein synthesis from UAG using a mutant initiator tRNA allows tightly regulated expression of specific genes. This may be generally useful for overproduction in E. coli and other eubacteria of proteins which are toxic to these cells.

Characterization of the fluorescent bimane derivative of E. coli initiator transfer RNA (tRNA fMet)

The invariant modified base 4-thiouridine of the E. Coli initiator tRNA was chemically modified using a sulfhydryl specific fluorogenic probe, monobromobimane. The modified tRNAfMet is virtually indistinguishable biochemically from the native form in the aminoacylation and formylation reactions, and in its binding behavior to the ribosomal P site. Fluorescence quenching by I- increases 40% when the modified tRNA is charged with formylmethionine, even at this relatively well-shielded position in the tRNA elbow. Most important, the fluorescence polarization increases by a factor of 2, to almost the irrotational value, when fMet-tRNAfMet binds to the ribosomal P site, providing a useful tool for studying fMet-tRNAfMet-ribosome interaction equilibria and kinetics.

Antico-operative binding of bacterial and mammalian initiator tRNA Met to methionyl-tRNA synthetase from Escherichia coli

The reaction scheme of methionyl-tRNA synthetase from Escherichia coli with the initiator tRNA s Met from E. coli and rabbit liver, respectively, has been resolved. The statistical rate constants for the formation, k R, and for the dissociation, k D, of the 1:1 complex of these tRNAs with the dimeric enzyme have been calculated. Identical k R values of 250 μ m −1 s −1 reflect similar behaviour for antico-operative binding of both tRNA s Met to native methionyl-tRNA synthetase. Advantage was taken of the difference in extent of tryptophan fluorescence-quenching induced by the bacterial and mammalian initiator tRNA s Met to measure the mode of exchange of these tRNAs antico-operatively bound to the enzyme. Analysis of the results reveals that antico-operativity does not arise from structural asymmetric assembly of the enzyme subunits. Indeed, both subunits can potentially bind a tRNA molecule. Exchange between tRNA molecules can occur via a transient complex in which both sites are occupied. Either strong and weak sites reciprocate between subunits on the transient complex or occupation of the weak site induces symmetry of this complex. While in the present case, these two alternatives are kinetically indistinguishable, they do account for the observation that, upon increasing the concentration of the competing mammalian tRNA, the rate of exchange of the E. coli initiator tRNA Met is enhanced, due to its faster rate of dissociation from the transient complex. Finally, it has been verified that in the case of the trypsin-modified methionyl-tRNA synthetase which cannot provide more than one binding site for tRNA, exchange of enzymebound bacterial tRNA by mammalian tRNA does proceed to a limiting rate independent of the mammalian tRNA concentration present in the solution.

Selective modification of cytidine, undine, guanosine and pseudouridine residues in Escherichia coli leucine transfer ribonucleic acid

The specificity of methoxyamine for the cytidine residues in an Escherichia coli leuoine transfer RNA (tRNA 1 leu is described in detail. Of the six non-hydrogen-bonded cytidine residues in the clover-leaf model of this tRNA, four are very reactive (C-35, 53, 85 and 86) and two are unreactive (C-67 and 79). The specificity of l-cyclohexyl-3-[2-morpholino-(4)-ethyl]carbodiimide methotosylate for the uridine, guanosine and pseudouridine residues in the leucine tRNA was also investigated. The carbodiimide completely modified four uridine residues (U-33, 34, 50 and 51) and partially modified G-37 and Ψ-39. For technical reasons, the sites of partial modification in loop I of the tRNA were difficult to establish. There was no modification of base residues in loop IV nor of U-59 at the base of stem e of the tRNA. The modification patterns described for the leucine tRNA are compared with those observed for the E. coli initiator tRNA 1 met and su + III tyrosine tRNA. Several general conclusions regarding tRNA conformation are made. In particular, the evidence supporting a diversity of anticodon loop structures amongst tRNAs is discussed.

Specific codon-anticodon interaction of an initiator-tRNA fragment.

WE have studied the relationship between structure and function of a tRNA by investigating the behaviour of two structurally defined fragments of the molecule. We first describe the properties of an isolated nonadecanucleotide fragment from the E. coli initiator tRNA, tRNAFmet. We show that this fragment, which contains the proposed anticodon CAU and a segment of paired bases, is specifically bound to ribosomes in the presence of the initiator triplets, ApUpG and GpUpG (refs. 1 and 2). We then show that an undecanucleotide fragment, which contains the anticodon but not a segment of paired bases, is not so bound.