The fate of norleucine as a replacement for methionine in protein synthesis.

In vivo synthesised protein with norleucine occupying one half of the normal methionine loci was prepared using a methionine auxotroph of Escherichia coli K12. The extent of charging of the analogue onto both tRNA_met species and subsequent incorporation into soluble protein was monitored with a double-labelling system comprising [G-³H]norleucine and [³⁵S]methionine. Further experiments established that norleucine can be formylated in vivo once charged onto the initiator tRNA^f_met. An N-terminal analysis of the crude soluble protein revealed that formylnorleucyl-tRNA^f_met can initiate protein synthesis and that the formyl group is then removed from the nascent polypeptide. We were also led to conclude that the N-terminal methionine-amino peptidase does not recognise the analogue in this position. Slow growth rates on the methionine analogue have been partly attributed to limiting levels of charged tRNA^m_met, resulting in turn from the inefficiency of norleucine charging by methionyl-tRNA synthetase. Finally no evidence has been found for the production of aberrant protein as a result of norleucine incorporation, implying that limited growth on the analogue is due to its inability to replace methionine as the precursor of S-adenosyl methionine.     

Escherichia coli formylmethionine tRNA: methylation of specific guanine and adenine residues catalyzed by HeLa cells tRNA methylases and the effect of these methylations on its biological properties

Purified HeLa cell tRNA methylases have been used for site-specific methylations of Escherichia coli formylmethionine transfer ribonucleic acid (tRNA^fMet). Guanine-N²-methylase catalyzed the methylation of a specific guanine residue (G₂₇) and adenine-1-methylase that of a specific adenine residue (A₅₉). The combined action of both of these enzymes leads to a total incorporation of two methyl groups and results in the methylation of both G₂₇ and A₅₉.The effect of introducing additional methyl groups on the function of tRNA has been studied by a comparison in vitro of the biological properties of tRNA^fMet and enzymically methylated tRNA^fMet. It was found that none of the following properties of E. coli tRNA^fMet are altered to any significant extent by methylation: (a) rate, extent, and specificity of aminoacylation, (b) ability of methionyl-tRNA to be enzymically formylated, and (c) ability of formylmethionyl-tRNA to initiate protein synthesis in cell-free extracts of E. coli in the presence of f2 RNA as messenger. Also, the temperature versus absorbance profile of the doubly methylated tRNA^fmet was virtually identical to that of the E. coli tRNA^fMet, and enzymically methylated tRNA^fmet resembled tRNA^fMet in that both were resistant to deacylation by E. coli, N-acylaminoacyl-tRNA hydrolase.     

Separation and comparison of primary structures of three formylmethionine tRNAs from E. coli K-12 MO

Three chromatographically distinct tRNAs^fMet from $\text{E. coli}$ K-12 MO were separated by reversed-phase chromatography and designated tRNA_A^fMet, tRNA_B^fMet, and tRNA₃^fMet. The tRNA_A^fMet corresponds to the published sequence for tRNA^fMet ($\text{E. coli}$). The tRNA_B^fMet differs from tRNA_A^fMet in that the 4-thiouridine in nucleotide position 8 has interacted with cytidine in position 13 to form a cross-linked product. The tRNA₃^fMet differs from tRNA_A^fMet in that 7-methyl-guanosine (in position 47) has been replaced by adenosine.     

Transfer RNA-mediated antitermination in vitro

The threonyl-tRNA synthetase gene (thrS) is a member of the T-box family of ~250 genes, found essentially in Gram-positive bacteria, regulated by a tRNA-dependent antitermination mechanism in response to starvation for the cognate amino acid. While interaction between uncharged tRNA and the untranslated leader region of these genes has been firmly established by genetic means, attempts to show this interaction or to reconstitute the antitermination mechanism in vitro using purified tRNAs have so far failed. In addition, a number of conserved sequences have been identified in the T-box leaders, for which no function has yet been assigned. This suggests that factors other than the tRNA are important for this type of control. Here we demonstrate tRNA-mediated antitermination for the first time in vitro, using the regulatory tRNA^Thr isoacceptor isolated from Bacillus subtilis and a partially purified protein fraction. As predicted by the model, aminoacylation of tRNA^Thr(GGU) with threonine completely abolishes its ability to act as an effector. The role of the partially purified protein fraction can be functionally substituted by high concentrations of spermidine. However, this polyamine does not play a significant role in the induction of thrS expression in vivo, suggesting that it is specific protein co-factors that promote T-box gene regulation in conjunction with uncharged tRNA.     

Crucial contribution of the multiple copies of the initiator tRNA genes in the fidelity of tRNAfMet selection on the ribosomal P-site in Escherichia coli

The accuracy of the initiator tRNA (tRNA^fMet) selection in the ribosomal P-site is central to the fidelity of protein synthesis. A highly conserved occurrence of three consecutive G–C base pairs in the anticodon stem of tRNA^fMet contributes to its preferential selection in the P-site. In a genetic screen, using a plasmid borne copy of an inactive tRNA^fMet mutant wherein the three G–C base pairs were changed, we isolated Escherichia coli strains that allow efficient initiation with the tRNA^fMet mutant. Here, extensive characterization of two such strains revealed novel mutations in the metZWV promoter severely compromising tRNA^fMet levels. Low cellular abundance of the chromosomally encoded tRNA^fMet allows efficient initiation with the tRNA^fMet mutant and an elongator tRNA^Gln, revealing that a high abundance of the cellular tRNA^fMet is crucial for the fidelity of initiator tRNA selection on the ribosomal P-site in E. coli. We discuss possible implications of the changes in the cellular tRNA^fMet abundance in proteome remodeling.     

Isolation and some properties of DNA coding for tRNA1met from Xenopus laevis

DNA containing the reiterated genes for tRNA₁^met has been partially purified from Xenopus laevis by centrifugation in actinomycin C₁-CsCl and Ag⁺-Cs₂SO₄ gradients. These gradients separate the tRNA₁^met genes from those coding for tRNA₂^met and tRNA^val, thus confirming our earlier suggestion that these genes are not intermingled with each other (Clarkson, Birnstiel, and Purdom, 1973). The gradients also demonstrate the existence of a minor 5S DNA fraction which appears to differ from that previously isolated by Brown, Wensink, and Jordan (1971).When the enriched tDNA₁^met is digested to completion with either of the restriction endonucleases EcoRI or Hpa I, the tRNA₁^met genes are predominantly found within DNA fragments that are about 3100 base pairs long. A partial digestion with EcoRI shows that these fragments arise from the regular spacing of the enzyme restriction sites. The 3100 base pair EcoRI fragments are cleaved by Hpa I into fragments of two size classes, one of which is about 2200 base pairs long and contains the tRNA₁^met genes. The shorter fragments are about 700 base pairs long, and they appear to contain genes coding for at least one other kind of tRNA species. X. laevis tDNA₁^met thus comprises tandemly repeated DNA whose component parts show little if any length heterogeneity.     

Structure and function of Escherichia coli formylmethionine transfer RNA : II. Effect of modification of guanosine residues on aminoacyl synthetase recognition

Guanosine residues in Escherichia coli formylmethionine transfer RNA have been modified by photo-oxidation in the presence of methylene blue. After irradiation to the extent of 50% loss of amino-acid acceptor activity, separation of active and inactive molecules has shown that guanosine rsesidues in the stem adjacent to the dihydrouridine loop and the guanosine at position no. 2 from the 5′ terminus are not required for aminoacylation or transformylation. Active molecules containing these modifications are amino-acylated with a K_m threefold higher than that for unmodified tRNA^fMet. No change in V_max occurs. Modification of a guanosine residue in the small loop joining the anticodon stem and the TΨC stem results in inactivation of methionine acceptor activity. This represents the first experimental evidence that a modification in this region affects the aminoacylation of a tRNA.     

Sequence organization of a cloned tDNA1met fragment from xenopus laevis

3.18 kb fragments of X. laevis DNA coding for tRNA₁^met have been inserted into a λ vector via Hind III termini and cloned in E. coli. The organization of one cloned fragment has been analyzed by restriction endonuclease digestion and RNA-DNA hybridization. From the distribution of sites for three enzymes, this fragment appears to be typical of the majority of λ. laevis tandem tDNA₁^met repeat units. Evidence is presented to suggest that it contains two genes coding for tRNA₁^met and at least one gene coding for a second as yet unidentified 4S RNA species. The two tRNA₁^met genes are located on the same DNA strand 0.96 and 1.38 kb from one end of the repeat unit. A detailed restriction map for 19 enzymes reveals that the spacers between these genes are not identical, and it provides no indication of short repetitive sequence elements within the spacers.     

Codon-Anticodon Recognition in the Bacillus subtilis glyQS T Box Riboswitch: RNA-DEPENDENT CODON SELECTION OUTSIDE THE RIBOSOME*

Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the “Specifier Sequence,” in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNA^Gly anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3′ of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system.     

A simplified method for study of RNA conformation--reaction of formylmethionine transfer RNA with [14C]methylamine-bisulfite

A new chemical method for radioactive labeling of single-stranded regions of RNA has been used to probe the three-dimensional structure of E. coli tRNA^fMet in solution. The procedure involves conversion of cytosine residues to N⁴-[¹⁴C]methylcytosines by treatment with ¹⁴CH₃NH₂ and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces ¹⁴C-labeled oligonucleotides which comigrate with the corresponding unlabeled oligonucleotides, facilitating structural analysis. By this procedure, E. coli tRNA^fMet has been found to contain only six reactive cytosines: C₁, C₁₆, C₁₇, C₃₅, C₇₅ and C₇₆. In addition, slow reaction at C^m₃₃ was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNA^fMet obtained by two other chemical methods. The methylamine-bisulfite procedure is recommended for studying the ordered structure of more complex polyribonucleotides such as viral and ribosomal RNAs.     

tRNA methylases from HeLa cells: purification and properties of an adenine-1-methylase and a guanine-N2-methylase

Two enzymes (methylases) that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to tRNA (prepared from Escherichia coli) have been partially purified from extracts of HeLa cells. One catalyzes the methylation of adenine residues of the tRNA to give 1-methyladenine units and the other is responsible for the conversion of guanine residues to N²-methylguanine and N²,N²-dimethylguanine (and may be a mixture of two enzymes). Activities of these relatively unstable enzymes could be maintained by storage at ?20 °C in the presence of 50% glycerol. Substrate specificity studies have revealed that bacterial tRNA (E. coli, Bacillus subtilis) can be used as substrate, whereas tRNA of animal origin (HeLa cells, rat liver) cannot be used. Of the specific tRNA's tested, E. coli tRNA^fMet was used as substrate by both enzymes. E. coli tRNA^Tyr was used by the adenine-1-methylase but not by the guanine-N²-methylase. The adenine-1-methylase catalyzed the transfer of approximately one methyl group per mole of either tRNA^fMet or tRNA^Tyr offered as substrate; in the presence of the guanine-N²-methylase 1 mole of E. coli tRNA^fMet accepted 1 mole of methyl. Studies with the use of both enzymes established that enzymic methylation of the guanine site of E. coli tRNA^fMet did not interfere with subsequent methylation of an adenine residue and neither did prior methylation of adenine interfere with the subsequent methylation of a guanine residue. In the presence of both enzymes, approximately 2 moles of methyl groups were accepted by 1 mole of the E. coli tRNA^fMet.     

Effects of dilute HCl on yeast tRNAPhe and E. coli tRNAfMet1

HCl treatment of yeast tRNA^Phe under conditions generally used for excision of `Y' base results in structure and conformation changes as monitored by line widths in the PMR spectra at 220 MHz and by optical rotation. Like exposure of E. coli tRNA^fMet₁ causes similar changes in the PMR spectra and optical rotation although no residues are eliminated. Electrophoresis in polyacrylamide gels provides evidence for aggregation in HCl-treated tRNA^fMet₁. One must thus consider a general effect of HCl exposure as well as possible residue removal in assessing induced structural and conformation changes in tRNA.     

Combination of the Loss of cmnmU34 with the Lack of sU34 Modifications of tRNA, tRNA, and tRNA Altered Mitochondrial Biogenesis and Respiration

Yeast Saccharomyces cerevisiae MTO2, MTO1, and MSS1 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of carboxymethylaminomethyl (cmnm)⁵s²U₃₄ in mitochondrial tRNA^Lys, tRNA^Glu, and tRNA^Gln. In fact, Mto1p and Mss1p are involved in the biosynthesis of the cmnm⁵ group (cmnm⁵U₃₄), while Mto2p is responsible for the 2-thiouridylation (s²U₃₄) of these tRNAs. Previous studies showed that partial modifications at U₃₄ in mitochondrial tRNA enabled mto1, mto2, and mss1 strains to respire. In this report, we investigated the functional interaction between MTO2, MTO1, and MSS1 genes by using the mto2, mto1, and mss1 single, double, and triple mutants. Strikingly, the deletion of MTO2 was synthetically lethal with a mutation of MSS1 or deletion of MTO1 on medium containing glycerol but not on medium containing glucose. Interestingly, there were no detectable levels of nine tRNAs including tRNA^Lys, tRNA^Glu, and tRNA^Gln in mto2/mss1, mto2/mto1, and mto2/mto1/mss1 strains. Furthermore, mto2/mss1, mto2/mto1, and mto2/mto1/mss1 mutants exhibited extremely low levels of COX1 and CYTB mRNA and 15S and 21S rRNA as well as the complete loss of mitochondrial protein synthesis. The synthetic enhancement combinations likely resulted from the completely abolished modification at U₃₄ of tRNA^Lys, tRNA^Glu, and tRNA^Gln, caused by the combination of eliminating the 2-thiouridylation by the mto2 mutation with the absence of the cmnm⁵U₃₄ by the mto1 or mss1 mutation. The complete loss of modifications at U₃₄ of tRNAs altered mitochondrial RNA metabolisms, causing a degradation of mitochondrial tRNA, mRNA, and rRNAs. As a result, failures in mitochondrial RNA metabolisms were responsible for the complete loss of mitochondrial translation. Consequently, defects in mitochondrial protein synthesis caused the instability of their mitochondrial genomes, thus producing the respiratory-deficient phenotypes. Therefore, our findings demonstrated a critical role of modifications at U₃₄ of tRNA^Lys, tRNA^Glu, and tRNA^Gln in maintenance of mitochondrial genome, mitochondrial RNA stability, translation, and respiratory function.     

The preparation and properties of a stable mercury derivative of transfer RNAs from Escherichia coli

Further investigations into the properties of the mercury derivative formed by the reaction of 4-thiouridine-containing tRNAs and pentafluorophenylmercury chloride have been carried out. tRNA^fMet (which contains only one 4-thiouridine residue) has been isolated by a one-step column Chromatographic procedure from unfractionated Escherichia coli tRNA and has been shown to react with the mercury compound to give a derivative which has similar properties to those previously reported for the corresponding mercury derivative of tRNA^Tyr which contains two adjacent 4-thiouridine residues. The mercury derivative of tRNA^Tyr appears to be a competitive inhibitor of tRNA^Tyr in the aminoacylation reaction (tRNA^TyrK_m = 0.42 μM, mercury derivative of tRNA^TyrK_i = 0.11 μM). The mercury derivative of Tyr-tRNA^Tyr can be made, but only by the reaction of the mercury compound with the aminoacylated tRNA.     

Effects of Regulatory Mutations upon Methionine Biosynthesis in Saccharomyces cerevisiae: Loci eth2-eth3-eth10

The effects of mutations occurring at three independent loci, eth2, eth3, and eth10, were studied on the basis of several criteria: level of resistance towards two methionine analogues (ethionine and selenomethionine), pool sizes of free methionine and S-adenosyl methionine (SAM) under different growth conditions, and susceptibility towards methionine-mediated repression and SAM-mediated repression of some enzymes involved in methionine biosynthesis (met group I enzymes). It was shown that: (i) the level of resistance towards both methionine analogues roughly correlates with the amount of methionine accumulated in the pool; (ii) the repressibility of met group I enzymes by exogenous methionine is either abolished or greatly lowered, depending upon the mutation studied; (iii) the repressibility of the same enzymes by exogenous SAM remains, in at least three mutants studied, close to that observed in a wild-type strain; (iv) the accumulation of SAM does not occur in the most extreme mutants either from endogenously overproduced or from exogenously supplied methionine: (v) the two methionine-activating enzymes, methionyl-transfer ribonucleic acid (tRNA) synthetase and methionine adenosyl transferase, do not seem modified in any of the mutants presented here; and (vi) the amount of tRNA^met and its level of charging are alike in all strains. Thus, the three recessive mutations presented here affect methionine-mediated repression, both at the level of overall methionine biosynthesis which results in its accumulation in the pool, and at the level of the synthesis of met group I enzymes. The implications of these findings are discussed.