Glycine codon discrimination and the nucleotide in position 32 of the anticodon loop.

Using an in vitro protein-synthesizing system that allowed us to monitor separately the reading of each glycine codon, we have previously shown, that in constructs based on glycine tRNA1 from Escherichia coli the nature of the nucleotide in position 32 determines the ability of the anticodon UCC to discriminate between the glycine codons. Thus, with a U in position 32 the anticodon UCC discriminated according to the wobble rules, but with a C in this position it had lost its ability to discriminate. In the present paper we show that the same is true also for constructs based on mycoplasma glycine tRNA.

The nucleotide in position 32 of the tRNA anticodon loop determines ability of anticodon UCC to discriminate among glycine codons.

We have investigated the influence of structures in the tRNA anticodon loop and stem on the ability of the anticodon to discriminate among codons. We had previously shown that anticodon UCC, when placed in the structural context of tRNA(Gly1) from Escherichia coli, discriminated efficiently between the glycine codons, as required by the wobble rules. Thus, this anticodon read GGA and GGG but did not read GGU and GGC, whereas in mycoplasma tRNA(Gly), the same anticodon did not discriminate among the glycine codons.

Codon discrimination and anticodon structural context.

Site-directed mutagenesis has been used to change the nucleotide C in the wobble position of tRNA(1Gly) (CCC) to U. The mutated tRNA was tested for its ability to read glycine codons in an in vitro protein-synthesizing system programmed with the phage message MS2-RNA that had been modified by site-directed mutagenesis so as to make it possible to monitor conveniently the reading of all four glycine codons. The results showed that while the efficiency of tRNA(1Gly) (UCC) was comparable to that of mycoplasma tRNA(Gly) (UCC) in the reading of the codon GGA, the mycoplasma tRNA(Gly) was far more efficient than the tRNA(1Gly) (UCC) in the reading of the codons GGU and GGC.

Apparent lack of discrimination in the reading of certain codons in Mycoplasma mycoides.

We report a cluster of four tRNA genes from Mycoplasma mycoides as well as the sequence of the alanine, proline, and valine tRNAs and the serine tRNA reading the UCN codons (where N stands for G, A, C, or U). This brings the total number of tRNA genes that we have so far characterized in this organism to 14, 6 of which code for tRNAs that read the codons of family boxes. In each of these latter cases, we found only one gene per family box, and the gene sequence contains a thymidine in the position corresponding to the wobble nucleotide, with the exception of the arginine tRNA gene that has an adenosine in this position.

Unconventional reading of the glycine codons.

We have used a protein-synthesizing in vitro system programmed with the phage message MS2-RNA to investigate the ability of glycyl-tRNAs with different anticodons to read the glycine codons. Under conditions of no competition, when the glycyl-tRNA analyzed was the only source of glycine for protein synthesis, each of the isoacceptors tested, tRNA1Gly (anticodon CCC), tRNA2Gly (anticodon N/UCC), tRNA3Gly (anticodon GCC) from Escherichia coli, and tRNAGly (anticodon UCC) from Mycoplasma mycoides, could read all of the glycine codons in the MS2 coat protein cistron (GGU, GGC, GGA, and GGG). However, tRNA1Gly seemed to have difficulties reading through the whole cistron.

Codon reading and translational error. Reading of the glutamine and lysine codons during protein synthesis in vitro.

The reading of glutamine and lysine codons during protein synthesis in vitro has been investigated using an MS2-RNA-programed system derived from Escherichia coli. Under conditions when either glutaminyl-tRNA1Gln (s2UUG) or glutaminyl-tRNA2Gln (CUG) was the only source of glutamine for protein synthesis both tRNAs were able to read the glutamine codons CAA and CAG as indicated by the incorporation of labeled glutamine into the pertinent coat protein tryptic peptides. On the other hand, when the two glutamine tRNAs competed for the codon CAA the reading efficiency of the anticodon s2UUG, which reads the codon according to the wobble rules, was almost 40 times higher than that of the competing anticodon CUG, which reads the codon by "two out of three," i.

Aberrations of the classic codon reading scheme during protein synthesis in vitro.

Using a protein synthesizing in vitro system programmed with MS2-RNA, the ability of alanine tRNAs with the anticodons U*GC (U* represents 5-oxyacetic acid uridine monophosphate) and IGC to read the alanine codons in the coat protein cistron of MS2 has been determined both under conditions of no competition, where the alanyl-tRNA used was the only aminoacylated tRNAAla present in the system, and in experiments where the two alanyl-tRNAs were competing against each other. Under conditions of no competition, each of the anticodons can read all four alanine codons. However, when the anticodons compete for the codon GCC, the anticodon IGC, which can read all three positions of the codon according to the rules of Watson-Crick base pairing, is considerably more efficient than U*GC, which misreads the codon by reading only the first two positions and presumably disregards the third nucleotide of the codon.

Relative efficiency of anticodons in reading the valine codons during protein synthesis in vitro.

Using a protein synthesizing in vitro system programmed with MS 2-RNA, the relative efficiency (in the presence of each other) of valine tRNAs with the anticodons U*AC (U* represents 5-oxyacetic acid uridine monophosphate), GAC, and IAC to read the valine codons was investigated. An anticodon which can read all three positions of the codon according to the rules of Watson-Crick base-pairing and the wobble hypothesis is an order of magnitude more efficient than an anticodon which misreads the codon by reading only the first two positions and presumably disregards the third nucleotide of the codon. There are two seeming exceptions to this behavior: the anticodon U*AC reads the codon GUU quite efficiently and IAC is as effective as U*AC in reading the codon GUG.

"Two out of three": an alternative method for codon reading.

An alternative method for codon reading, whereby only the first two codon nucleotides are recognized by the anticodon, is discussed and the experimental evidence for this "two of three" reading method is reviewed. Misreading of codons by the "two out of three" method could pose a significant threat to the fidelity of protein synthesis unless the genetic code is organized in such a way as to prevent this method from being used when it might compromise translational fidelity. Inspection of the genetic code shows that it is arranged in such a way that the "two out of three" reading method can be used without translational errors.

Aminoacyl adenylate, a normal intermediate or a dead end in aminoacylation of transfer ribonucleic acid.

The shape of the time curve for the aminoacylation of tRNA has been investigated using five different amino acid:tRNA ligases. Four of these enzymes showed a lag in the time curve during the early phase of the first catalytic turnover of the enzyme. In each case, the lag period could be abolished by preincubating the ligase with amino acid, ATP, and Mg2+ under conditions known to give an aminoacyl adenylate-enzyme complex.

Codon-acticodon recognition in the valine codon family.

An in vitro protein-synthesizing system completely dependent on added valine tRNA (valyl-tRNAval) and programmed with RNA from the phage MS2 has been used to investigate the incorporation into MS2 coat protein of valine from isoaccepting valyl-tRNAsval with the anticodons U AC (U represents 5-oxyacetic acid uridine monophosphate), GAC, and IAC in response to the four valine codons GUU, GUC, GUA, and GUG. By examining the incorporation of valine into NH2-terminal and internal positions of three tryptic peptides from the MS2 coat protein it has been established that these anticodons each recognize all four valine codons. We therefore conclude that under our conditions of in vitro protein synthesis the genetic code, as far as the valine codons are concerned, is operationally a two letter code, i.