Translation in vitro of codon UGA as tryptophan in Mycoplasma capricolum.

The in-frame UGA codons in the synthetic messenger RNA were translated in the cell-free system of Mycoplasma capricolum. The result, together with the occurrence of codon UGA at tryptophan sites in the genes and the presence of tRNA(UCATrp) pairing with UGA, clearly indicated that UGA is a tryptophan codon in this bacterium.     

Unconventional codon reading by Mycoplasma mycoides tRNAs as revealed by partial sequence analysis.

Continuing our investigation of the tRNA genes and gene products in Mycoplasma mycoides, we report the sequence of the gene for tRNALeu (CAA) as well as partial primary structures of the following tRNAs: Leu (CAA), Leu (UAG), Arg (UCU), Thr (AGU) and Ile (CAU). It is suggested that in M. mycoides, at least some of the family codon boxes are read by only one tRNA each, using an unconventional method which does not discriminate between the nucleotides in the third codon position. M. mycoides is the first free-living organism known to use an unconventional method of this kind.     

The phenylalanine tRNA from Mycoplasma sp. (Kid): a tRNA lacking hypermodified nucleosides functional in protein synthesis

Phenylalanine tRNA from Mycoplasma sp. (Kid) was purified and characterized. The tRNA can be aminoacylated by phenylalanyl-tRNA synthetase from both Mycoplasma and E. coli. In a tRNA-dependent cell-free E. coli amino acid incorporating system programmed with poly U pure Mycoplasma tRNA(Phe) was fully active in promoting phenylalanine incorporation, even in direct competition with homologous E. coli tRNA(Phe). Since the Mycoplasma tRNA lacks isopentenyladenosine, or any related hypermodified nucleoside, it appears that the presence of such nucleosides in tRNA is not an absolute requirement for protein synthesis.     

Codon recognition patterns as deduced from sequences of the complete set of transfer RNA species in Mycoplasma capricolum. Resemblance to mitochondria

The nucleotide sequences of the complete set of tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been determined. This bacterium represents the first genetic system in which the sequences of all the tRNA species have been determined at the RNA level. There are 29 tRNA species: three for Leu, two each for Arg, Ile, Lys, Met, Ser, Thr and Trp, and one each for the other 12 amino acids as judged from aminoacylation and the anticodon nucleotide sequences. The number of tRNA species is the smallest among all known genetic systems except for mitochondria. The tRNA anticodon sequences have revealed several features characteristic of M. capricolum. (1) There is only one tRNA species each for Ala, Gly, Leu, Pro, Ser and Val family boxes (4-codon boxes), and these tRNAs all have an unmodified U residue at the first position of the anticodon. (2) There are two tRNAThr species having anticodons UGU and AGU; the first positions of these anticodons are unmodified. (3) There is only one tRNA with anticodon ICG in the Arg family box (CGN); this tRNA can translate codons CGU, CGC and CGA. No tRNA capable of translating codon CGG has been detected, suggesting that CGG is an unassigned codon in this bacterium. (4) A tRNATrp with anticodon UCA is present, and reads codon UGA as Trp. On the basis of these and other observations, novel codon recognition patterns in M. capricolum are proposed. A comparatively small total, 13, of modified nucleosides is contained in all M. capricolum tRNAs. The 5' end nucleoside of the T psi C-loop (position 54) of all tRNAs is uridine, not modified to ribothymidine. The anticodon composition, and hence codon recognition patterns, of M. capricolum tRNAs resemble those of mitochondrial tRNAs.     

The nucleotide sequence of formylmethionine tRNA from Mycoplasma mycoides sp. capri.

The nucleotide sequence of Mycoplasma mycoides sp. capri PG3 formylmethionine tRNA has been determined, using in vitro labeling techniques, to be pC-G-C-G-G-G-G-s4U-A-G-A-G-C-A-G-U-D (U)-G-G-D-A-G-C-U-C-G-C-C-G-G-G-C-U-C-A-U-A-A-C-C-C-G-G-A-G-G-C-C-G-C-A-G-G-U-psi- C-G-A-G-U-C-C-U-G-C-C-C-C-C-G-C-A-A-C-C-AOH. This tRNA contains only three modified nucleosides s4U, D and psi, all of which are derived from uridine. Both in the structural features which distinguish eukaryotic from prokaryotic initiator RNAs and in the overall sequence, this tRNA resembles a typical prokaryotic initiator tRNA. A comparison of the sequence of this tRNA with those of other prokaryotic initiator tRNAs suggests that taxonomically the Mycoplasma may be less related to the Cyanophyta (Anacystis nidulans) than to the bacteria and less related to the Enterobacteriaceae (Escherichia coli) than to the Bacillaceae (Bacillus subtilis).     

Selection of aminoacyl-tRNAs at sense codons: the size of the tRNA variable loop determines whether the immediate 3' nucleotide to the codon has a context effect.

Codon context can affect translational efficiency by several molecular mechanisms. The base stacking interactions between a codon-anticodon complex and the neighboring nucleotide immediately 3' can facilitate translation by amber suppressors and the tRNA structure is also known to modulate the sensitivity to context. In this study the relative rates of aminoacyl-tRNA selection were measured at four sense codons (UGG, CUC, UUC and UCA), in all four 3' nucleotide contexts, through direct competition with a programmed frameshift at a site derived from the release factor 2 gene. Two codons (UGG and UUC) are read by tRNAs with small variable regions and their rates of aminoacyl-tRNA selection correlated with the potential base stacking strength of the 3' neighboring nucleotide. The other two codons (CUC and UCA) are read by tRNAs with large variable regions and the rate of selection of the aminoacyl-tRNAs in these cases varied little among the four contexts. Re-examination of published data on amber suppression also revealed an inverse correlation between context sensitivity and the size of the variable region. Collectively the data suggest that a large variable loop in a tRNA decreases the influence of the 3' context on tRNA selection, probably by strengthening tRNA-ribosomal interactions.     

Selenocysteine, a highly specific component of certain enzymes, is incorporated by a UGA-directed co-translational mechanism

The opal termination codon UGA is used in both prokaryotic and eukaryotic species to direct the specific insertion of selenocysteine into certain selenium-dependent enzymes. So far a formate dehydrogenase (hydrogenase-linked) of Escherichia coli and glutathione peroxidases of murine, human and rat origin have been identified as enzymes containing selenocysteine residues encoded by UGA. A novel seryl-tRNA, anticodon UCA, that specifically recognizes the UGA codon is required for selenocysteine incorporation into formate dehydrogenase. A eukaryotic UGA suppressor tRNA with UCA anticodon that accepts serine and is phosphorylated to O-phosphoseryl-tRNA may have a corresponding function in glutathione peroxidase synthesis. Other factors required for the unusual usage of the in-frame UGA codons to specify selenocysteine incorporation and the biochemical mechanism involved in distinguishing these from normal UGA termination codons are discussed.     

Sequence studies of nonradioactive Mycoplasma tRNAPhe with the aid of polynucleotide phosphorylase and polynucleotide kinase

The known methods of enzymatic phosphorylation with [(32)P]phosphate of the 3'- or 5'-hydroxyl group of an oligonucleotide have been applied to oligonucleotides derived from Mycoplasma tRNA(Phe). The fingerprints obtained by both methods are very similar to each other and to that of uniformly labelled tRNA. The sequence of some oligonucleotides was determined by partial digestion of the 3'-phosphorylated fragment with spleen phosphodiesterase and of the corresponding 5'-phosphorylated fragment with venom phosphodiesterase.     

Mycoplasma genitalium and Mycoplasma pneumoniae share a 67 kilodalton protein as a main cross-reactive antigen

It was demonstrated that a 67 kilodalton (kDa) protein of Mycoplasma pneumoniae is a main cross-reactive antigen with similar molecular weight protein of Mycoplasma genitalium by Western blot analysis using monoclonal antibody to 67 kDa protein of M. pneumoniae and hyperimmune rabbit sera directed against each mycoplasma strain.     

Formylatable methionine transfer RNA from Mycoplasma: purification and comparison of partial nucleotide sequences with those of other prokaryotic initiator tRNAs.

The major species of the formylatable methionine tRNA from Mycoplasma mycoides var capri has been purified. The 5'- and 3'-terminal sequences of the purified tRNA are pC-G- and C-A-A-C-C-AOH, respectively. Thus, this tRNA also contains the unique structural feature found in two other prokaryotic initiator tRNAs in that the first nucleotide at the 5'-end cannot form a Watson-Crick type of base-pair to the fifth nucleotide from the 3'-end. The Mycoplasma tRNA does not contain ribothymidine; however, a specific uridine residue in the sequence G-U-psi-C-G- can be enzymatically methylated by E. coli extracts to yield G-T-psi-C-G. Since ribothymidine is absent in crude tRNA from this strain of Mycoplasma, the absence of T is probably due to the lack of a U yields T modifying enzyme.     

Selenocysteine synthase from Escherichia coli. Analysis of the reaction sequence

The product of the selA gene, selenocysteine synthase, is a pyridoxal 5-phosphate-containing enzyme which catalyzes the conversion of seryl-tRNA(Sec UCA) into selenocysteyl-tRNA(Sec UCA). Reduction of the aldimine group of pyridoxal 5-phosphate inactivates the enzyme. When reacted with seryl-tRNA(Sec UCA) as sole substrate, pyruvate (and possibly also ammonia) is released; in the presence of a high concentration of potassium borohydride, alanyl-tRNA(Sec UCA) is formed from seryl-tRNA(Sec UCA). These results support the notion that the formyl group of pyridoxal phosphate forms a Schiff base with the alpha-amino group of L-serine with the subsequent 2,3-elimination of a water molecule and the generation of an aminoacrylyl-tRNA(Sec UCA) intermediate. ATP is not required for this reaction step, but it is necessary for the conversion of aminoacrylyl-tRNA into selenocysteyl-tRNA(Sec UCA) which, in addition, requires the SELD protein and reduced selenium. Selenocysteine synthase forms a stable complex with seryl-tRNA(Sec UCA) with one tRNA molecule bound per two 50-kDa monomers. The enzyme does not interact with serine-inserting tRNA species. Taken together, the results show that biosynthesis of selenocysteine takes place in the enzyme-bound state and involves the dehydration of L-serine esterified to tRNA in a first step formally followed by the 2,3-addition of HSe- which is provided by the SELD protein in an ATP-dependent reaction in the form of a reactive selenium donor molecule.     

Interaction of a selenocysteine-incorporating tRNA with elongation factor Tu from E.coli.

Selenocysteine-incorporating tRNA(Sec)(UCA), the product of selC, was isolated from E.coli and aminoacylated with serine. The equilibrium dissociation constant for the interaction of Ser-tRNA(Sec)(UCA) with elongation factor Tu.GTP was determined to be 5.0 +/- 2.5 x 10(-8) M. Compared with the dissociation constants of the two elongator Ser-tRNA(Ser) species (Kd = 7 x 10(-10) M), the selenocysteine-incorporating UGA suppressor tRNA has an almost hundred fold weaker affinity for EF-Tu.GTP. This suggests a mechanism by which the Ser-tRNA(Sec) is prevented in recognition of UGA codons. This tRNA is not bound to EF-Tu.GTP and is converted to selenocysteinyl-tRNA(Sec). We also demonstrate the lack of an efficient interaction of Sec-tRNA(Sec)(UCA) with EF-Tu.GTP. The results of this work are in support of a mechanism by which the selenocysteine incorporation at UGA nonsense codons is mediated by an elongation factor other than EF-Tu.GTP.     

Codon reading scheme in Mycoplasma pneumoniae revealed by the analysis of the complete set of tRNA genes.

The 33 genes encoding the complete set of tRNA species in Mycoplasma pneumoniae have been cloned and sequenced. They are organized into 5 clusters in addition to 9 single genes. No redundant gene was found, indicating that 33 tRNAs correspond to 32 different anticodons and decode all 62 codons used in this organism. There is only one single tRNA for each of the Ala, Leu, Pro, and Val family boxes. Therefore, a simplified decoding system resembling that recently described for Mycoplasma capricolum (1) has to also exist in M.pneumoniae. However, analysis of the anticodon set and codon usage revealed features characteristic of the latter: (i) there is no obvious preference toward AT rich synonymous codons, (ii) CGG codons are assigned for arginine and are translated by tRNA Arg(UCG), and (iii) CNN or GNN anticodons are encountered in the Ser, Thr, Arg, and Gly family boxes. We thus propose that this codon-anticodon recognition pattern has emerged in the 'M.pneumoniae cluster' under a genomic economization strategy but without the influence of AT pressure.     

The nucleotide sequence of phenylalanine tRNA from Mycoplasma sp. (Kid)

The nucleotide sequence of Mycoplasma sp. (Kid) phenylalanine tRNA was determined to be pG-G-U-C-G-U-G-U-A-G-C-U-C-A-G-U-C-G-G-D-A-G-A-G-C-A-G-C- A-G-A-C-U-G-A-A-m(1)G-C-Psi-C-U-G-C-G-U-m(7)G-U-C-G-G-C-G-G-U-Psi-C-A-A-U-U-C-C-G-U-C-C-A-C-G-A-C-C-A-C-C-A(OH). It is characterized by the absence of ribothymidine and the presence of only few modified nucleotides.